Nanoarrays and methods of use thereof

ABSTRACT

Disclosed herein, inter alia, are nanoarrays and methods of use thereof.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.63/160,716, filed Mar. 12, 2021 and U.S. Provisional Application No.63/164,742, filed Mar. 23, 2021, which are incorporated herein byreference in their entirety and for all purposes.

BACKGROUND

Patterned arrays are an important tool in biomedical research, providinga two-dimensional platform that arranges biological samples and enableshigh-throughput analyses. Delivering breakthroughs in proteomics,multiplexed immunoassays, and complex genomic analyses, microarrays canbe designed to host thousands, or even ten-thousands, of features thatcan be subjected to simultaneous reaction conditions.

Next generation sequencing (NGS) methodologies make use ofsimultaneously sequencing millions of fragments of nucleic acids in asingle experiment. For example, sequencing-by-synthesis (SBS) istypically performed by imaging clusters of amplicons, referred to asfeatures, having multiple identical copies of a starting molecule. Inorder to maximize the rate of output of sequencing information, effortshave been made to increase the ratio of nucleotides sequenced per image.Miniaturization is required for increasing the scale and density of theamplicons, that could result in lower reagent consumption and fasterdata acquisitions, however greater challenges arise as the featuredimensions approach submicron domains. Disclosed herein, inter alia, aresolutions to these and other problems in the art.

BRIEF SUMMARY

In an aspect is provided an array, including: a solid support includinga surface, the surface comprising a plurality of wells separated fromeach other by interstitial regions on the surface, wherein one or morewells include a particle, wherein the particle includes a plurality ofbioconjugate reactive moieties, a plurality of oligonucleotide moieties,or a combination thereof; and wherein there is one particle per well.

In an aspect is provided a method of amplifying a target polynucleotide,the method including: contacting the array as described herein,including embodiments, with a plurality of oligonucleotide moieties,each oligonucleotide moiety including a bioconjugate reactive moietythat reacts and forms a bioconjugate linker that covalently links theoligonucleotide moiety to the particle, contacting the array with asample including a target polynucleotide; and amplifying the targetpolynucleotide to produce an amplification product, wherein amplifyingincludes extension of the oligonucleotide moiety hybridized to thetarget polynucleotide.

In an aspect is provided a method of amplifying a target polynucleotide,the method including: contacting the array as described herein,including embodiments and wherein the particle includes a plurality ofoligonucleotide moieties, with a sample including a targetpolynucleotide; and amplifying the target polynucleotide to produce anamplification product, wherein amplifying includes extension of theoligonucleotide moiety hybridized to the target polynucleotide.

In an aspect is provided a nucleic acid sequencing device, including: astage configured to hold an array or solid support as described herein,including embodiments; an array or solid support as described herein,including embodiments; and a detector for obtaining sequencing data.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-ID. Illustrations of the different well shapes of the array.Using known nanolithographic fabrication techniques, a glass substratemay be etched such that the well is anisotropic (FIG. 1A), partiallyanisotropic (FIG. 1B), or isotropic (FIG. 1C). The array may include aresist polymer (e.g., a fluorinated polymer later) prior to receiving anadditional polymer coating (e.g., a poloxamer or alkoxysilyl polymer).The resist may be removed prior to the addition of the additionalpolymer using known techniques in the art (e.g., solvent removal). Inembodiments, the additional polymer coating reduces the non-specificbinding of oligonucleotide moieties. Alternatively, the wells may bedirectly formed within the resist (e.g., a nanoimprint resist) asdepicted in FIG. 1D, wherein the resist is attached to a glass solidsupport. In embodiments, the resist is not removed prior to loading theparticles.

FIGS. 2A-2B are illustrations of particles in wells of an array asdescribed herein. FIG. 2A illustrates particles decorated withbioconjugate reactive moieties, for example, azide-reactive species. Theparticles are localized in wells of an array, wherein the array includesa polymer coating, for example, a silane functionalized polyethyleneglycol (Si-PEG) copolymer or a silane functionalized poly(acrylamide)(Si-Pam) copolymer coating. FIG. 2B illustrates particles in wells of asimilar polymer-coated array, wherein the particles includeoligonucleotides linked to the bioconjugate reactive groups. Theparticles may include oligonucleotide moieties, or the oligonucleotidemoieties may be added following deposition of the particles. Theoligonucleotides may be capable of hybridizing to template nucleic acidsfor subsequent amplification and sequencing applications.

FIG. 3 illustrates one embodiment wherein polymeric particles asdescribed herein are introduced in an anti-solvent (e.g., ethanol) to apatterned substrate. In the presence of anti-solvent the volume of theparticles is reduced relative to the volume of the particles in water.Subsequently, the reaction conditions are changed such that an aqueoussolvent (e.g., water) is present, which swells the particles, causingthem to remain trapped in the wells. The particles may includeoligonucleotide moieties, or the oligonucleotide moieties may be addedfollowing deposition of the particles.

FIG. 4 is a scanning electron micrograph of a subsection of the arraywith particles arrayed in each well. Magnification of image is 10,000×.The scale bar denotes 4 μm. The center-to-center distance of each wellis approximately 1 μm.

FIGS. 5A-5B are fluorescent images of an array of the invention showingparticles loaded in wells, the particles decorated with oligonucleotideprimers that have been labeled with FAM-labeled complementaryoligonucleotides. The wells have a 1 μm center-to-center distance (FIG.5A) and a 1.4 μm center-to-center distance (FIG. 5B). The center circleis a reference fiducial marker useful in alignment for detectionapparatus.

FIGS. 6A-6B are fluorescent images of FAM-labeled clusters generatedfrom oligonucleotide primer-coated particles that were hybridized totemplate nucleic acids and amplified to generate an amplificationproduct on the particle. To quantify the amount of non-specific bindingin the interstitial space, the array contains a reference interstitialchannel in the form of a cross, i.e., a region of interstitial spacethat does not include any particles. FIG. 6A is an image of an arraysubstrate prepared without any polymer coating (i.e., the substrate doesnot contain a hydrophilic polymer) and highlights the non-specificcluster amplification in the interstitial space. FIG. 6A shows that anoligonucleotide primer may bind to the interstitial space and beamplified. FIG. 6B is an image of an array that was coated with apassivating polymer p[PEGMA-co-TESPM] prior to particle loading, whichresulted in a significant reduction in non-specific clusters present inthe interstitial space.

FIGS. 7A-7B. FIG. 7A an illustration of the topological differencesbetween a comb and brush polymer. In embodiments, the solid supportincludes a passivating polymer coating of a brush or comb polymer. FIG.7B shows an embodiment of an amphiphilic polymer, generated bypolymerizing PEGMA and TMSPM monomers, where n is an integer from 1 to100; n1 and n2 are integers from 1 to 1000, respectively. FIG. 7Bdepicts a subunit of a copolymer that includes [PEGMA]_(n1) and[TMSPM]_(n2), however it is understood that the copolymer may includeadditional repeating subunits, e.g.,[PEGMA]_(n1)-[TMSPM]_(n2)-[PEGMA]_(n1)-[TMSPM]_(n2) or-[TMSPM]_(n2)-[PEGMA]_(n1)-[TMSPM]_(n2) and the like. The ratio ofhydrophilic segments (e.g., PEGMA) to hydrophobic segments (e.g., TMSPM)is governed by modulating the reaction conditions (e.g., monomerconcentrations, pH, and/or temperature).

FIG. 8 is a brightfield image of pure polymeric particles (i.e.,particles not containing a solid particle core) synthesized as describedherein having an average diameter of about 550 nm. Exemplary particlesof the invention have a diameter of less than 1000 nm.

FIGS. 9A-9D depict images of a patterned solid support that includeswells separated by 1.4 um (center to center) filled with particles thatinclude a plurality of oligonucleotide moieties. The diameter of theparticle core was varied, from 400 nm (FIG. 9A), 450 nm (FIG. 9B), and500 nm (FIG. 9C) and a fluorescently labeled complementary probehybridized to the immobilized oligonucleotide was detected. Particlecores with an average diameter of about 400 nm provided uniformcoverage. Optimizing the loading conditions and particle concentrationsprovided a surface with approximately 92% coverage (e.g., over 92% ofthe wells have a particle), as shown in FIG. 9D. Each discrete spotrepresents a feature available for sequencing, as confirmed by thefluorescent probe.

FIGS. 10A-10B. FIG. 10A is an illustration of functionalized particleincluding a particle core (e.g., a silica core) and a particle shell(e.g., a polymer shell). The illustration depicts the polymer shell asdistinct polymeric chains with exaggerated separation for clarity,however it is understood that the polymer chains form a network thatcoat (i.e., surround) the surface of the particle core. The thickness ofthe polymer layer can be controlled by varying the reaction conditions(e.g., time, temperature, and concentration of the correspondingmonomers) to provide a thickness of about 50 nm to about 200 nm. FIG.10B illustrates an individual polymeric chain covalently attached to theparticle core. The polymer chain is a polyethylene glycol methacrylate(PEGMA) and glicydyl methacrylate azide (GMA azide, or GMA-Az) copolymerin a 2:1 ratio (i.e., having an ng of 3). Alternative ng copolymers werealso synthesized by varying the ratio of PEGMA:GMA-Az 5:1 (ng=6) or 8:1(ng=9), providing tunable parameters to control the density ofoligonucleotide, which corresponds to density of the resultingpolynucleotides.

FIG. 11 . An overview of the polymerization and particle loading isprovided in FIG. 11 . A particle core (e.g., a silica or metallicnanoparticle) is incubated with monomers under suitable polymerizationconditions and particle polymers grow on the surface of the core. Thefunctionalized particle now includes a plurality of bioconjugatereactive moieties (e.g., azido moieties). The functionalized particlemay be loaded onto a patterned solid support (e.g., a multiwellcontainer that optionally includes a passivating polymer as describedherein) and arranged into the wells. Oligonucleotide moieties containinga reactive bioconjugate moiety (e.g., a DBCO moiety) are allowed tocontact the particles, wherein the oligonucleotides reacts with thebioconjugate groups on the polymers and forms a bioconjugate linker,thereby covalently immobilizing the oligonucleotides to the particle.Alternatively, prior to loading into the patterned solid support,oligonucleotide moieties containing a reactive bioconjugate moiety areallowed to contact the particles and form a bioconjugate linker, therebycovalently immobilizing the oligonucleotides to the particle. Theoligo-containing particles are then loaded onto the multiwell containerthat does not include a passivating polymer. A particle loaded patternedflow cell may include about 10,000 to about 50,000, or about 25,000immobilized oligonucleotides per square micrometer. The pattered solidsupport is then ready for standard clustering (e.g., template seedingand amplification) protocols and/or subsequent detection (e.g.,sequencing).

DETAILED DESCRIPTION

The aspects and embodiments described herein relate to nanoarrays andmethods of making and using nanoarrays.

I. Definitions

The practice of the technology described herein will employ, unlessindicated specifically to the contrary, conventional methods ofchemistry, biochemistry, organic chemistry, molecular biology,microbiology, recombinant DNA techniques, genetics, immunology, and cellbiology that are within the skill of the art, many of which aredescribed below for the purpose of illustration. Examples of suchtechniques are available in the literature. See, e.g., Singleton et al.,DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY 2nd ed., J. Wiley &Sons (New York, N.Y. 1994); and Sambrook and Green, Molecular Cloning: ALaboratory Manual, 4th Edition (2012). Methods, devices and materialssimilar or equivalent to those described herein can be used in thepractice of this invention.

All patents, patent applications, articles and publications mentionedherein, both supra and infra, are hereby expressly incorporated hereinby reference in their entireties.

Unless defined otherwise herein, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. Various scientificdictionaries that include the terms included herein are well known andavailable to those in the art. Although any methods and materialssimilar or equivalent to those described herein find use in the practiceor testing of the disclosure, some preferred methods and materials aredescribed. Accordingly, the terms defined immediately below are morefully described by reference to the specification as a whole. It is tobe understood that this disclosure is not limited to the particularmethodology, protocols, and reagents described, as these may vary,depending upon the context in which they are used by those of skill inthe art. The following definitions are provided to facilitateunderstanding of certain terms used frequently herein and are not meantto limit the scope of the present disclosure.

As used herein, the singular terms “a”, “an”, and “the” include theplural reference unless the context clearly indicates otherwise.Reference throughout this specification to, for example, “oneembodiment”, “an embodiment”, “another embodiment”, “a particularembodiment”, “a related embodiment”, “a certain embodiment”, “anadditional embodiment”, or “a further embodiment” or combinationsthereof means that a particular feature, structure or characteristicdescribed in connection with the embodiment is included in at least oneembodiment of the present disclosure. Thus, the appearances of theforegoing phrases in various places throughout this specification arenot necessarily all referring to the same embodiment. Furthermore, theparticular features, structures, or characteristics may be combined inany suitable manner in one or more embodiments.

As used herein, the term “about” means a range of values including thespecified value, which a person of ordinary skill in the art wouldconsider reasonably similar to the specified value. In embodiments, theterm “about” means within a standard deviation using measurementsgenerally acceptable in the art. In embodiments, about means a rangeextending to +/−10% of the specified value. In embodiments, about meansthe specified value.

Throughout this specification, unless the context requires otherwise,the words “comprise”, “comprises” and “comprising” will be understood toimply the inclusion of a stated step or element or group of steps orelements but not the exclusion of any other step or element or group ofsteps or elements. By “consisting of” is meant including, and limitedto, whatever follows the phrase “consisting of” Thus, the phrase“consisting of” indicates that the listed elements are required ormandatory, and that no other elements may be present. By “consistingessentially of” is meant including any elements listed after the phrase,and limited to other elements that do not interfere with or contributeto the activity or action specified in the disclosure for the listedelements. Thus, the phrase “consisting essentially of” indicates thatthe listed elements are required or mandatory, but that no otherelements are optional and may or may not be present depending uponwhether or not they affect the activity or action of the listedelements.

The terms “attached,” “bind,” and “bound” as used herein are used inaccordance with their plain and ordinary meanings and refer to anassociation between atoms or molecules. The association can be direct orindirect. For example, attached molecules may be directly bound to oneanother, e.g., by a covalent bond or non-covalent bond (e.g.electrostatic interactions (e.g. ionic bond, hydrogen bond, halogenbond), van der Waals interactions (e.g. dipole-dipole, dipole-induceddipole, London dispersion), ring stacking (pi effects), hydrophobicinteractions and the like). As a further example, two molecules may bebound indirectly to one another by way of direct binding to one or moreintermediate molecules, thereby forming a complex.

As used herein, the term “complement,” as used herein, refers to anucleotide (e.g., RNA or DNA) or a sequence of nucleotides capable ofbase pairing with a complementary nucleotide or sequence of nucleotides.As described herein and commonly known in the art the complementary(matching) nucleotide of adenosine is thymidine and the complementary(matching) nucleotide of guanosine is cytosine. Thus, a complement mayinclude a sequence of nucleotides that base pair with correspondingcomplementary nucleotides of a second nucleic acid sequence. Thenucleotides of a complement may partially or completely match thenucleotides of the second nucleic acid sequence. Where the nucleotidesof the complement completely match each nucleotide of the second nucleicacid sequence, the complement forms base pairs with each nucleotide ofthe second nucleic acid sequence. Where the nucleotides of thecomplement partially match the nucleotides of the second nucleic acidsequence, only some of the nucleotides of the complement form base pairswith nucleotides of the second nucleic acid sequence. Examples ofcomplementary sequences include coding and non-coding sequences, whereinthe non-coding sequence contains complementary nucleotides to the codingsequence and thus forms the complement of the coding sequence. A furtherexample of complementary sequences are sense and antisense sequences,wherein the sense sequence contains complementary nucleotides to theantisense sequence and thus forms the complement of the antisensesequence. Another example of complementary sequences are a templatesequence and an amplicon sequence polymerized by a polymerase along thetemplate sequence.

As described herein, the complementarity of sequences may be partial, inwhich only some of the nucleic acids match according to base pairing, orcomplete, where all the nucleic acids match according to base pairing.Thus, two sequences that are complementary to each other, may have aspecified percentage of nucleotides that complement one another (e.g.,about 60%, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or higher complementarity over a specifiedregion). In embodiments, two sequences are complementary when they arecompletely complementary, having 100% complementarity. In embodiments,one or both sequences in a pair of complementary sequences form portionsof longer polynucleotides, which may or may not include additionalregions of complementarity.

As used herein, the term “hybridize” or “specifically hybridize” refersto a process where two complementary nucleic acid strands anneal to eachother under appropriately stringent conditions. Hybridizations aretypically and preferably conducted with oligonucleotides. Non-limitingexamples of nucleic acid hybridization techniques are described in, forexample, Sambrook, et al., Molecular Cloning: A Laboratory Manual (2ndEdition, 1989). Those skilled in the art understand how to estimate andadjust the stringency of hybridization conditions such that sequenceshaving at least a desired level of complementarity will stablyhybridize, while those having lower complementarity will not.Hybridization reactions can be performed under conditions of different“stringency”. For example, a low stringency hybridization reaction iscarried out at about 40° C. in 10×SSC. A moderate stringencyhybridization may be performed at about 50° C. in 6×SSC. A highstringency hybridization reaction is generally performed at about 60° C.in 1×SSC. Hybridization reactions can also be performed under“physiological conditions” which is well known to one of skill in theart (e.g., a physiological condition is the temperature, ionic strength,pH and concentration of Mg²⁺ normally found in vivo). The propensity forhybridization between nucleic acids depends on the temperature and ionicstrength of their milieu, the length of the nucleic acids and the degreeof complementarity. The effect of these parameters on hybridization isdescribed in, for example, Sambrook J., Fritsch E. F., Maniatis T.,Molecular cloning: a laboratory manual, Cold Spring Harbor LaboratoryPress, New York (1989). As used herein, hybridization of a primer, or ofa DNA extension product, respectively, is extendable by creation of aphosphodiester bond with an available nucleotide or nucleotide analoguecapable of forming a phosphodiester bond, therewith. For example,hybridization can be performed at a temperature ranging from 15° C. to95° C. In some embodiments, the hybridization is performed at atemperature of about 20° C., about 25° C., about 30° C., about 35° C.,about 40° C., about 45° C., about 50° C., about 55° C., about 60° C.,about 65° C., about 70° C., about 75° C., about 80° C., about 85° C.,about 90° C., or about 95° C. In other embodiments, the stringency ofthe hybridization can be further altered by the addition or removal ofcomponents of the buffered solution.

As used herein, “specifically hybridizes” refers to preferentialhybridization under hybridization conditions where two nucleic acids, orportions thereof, that are substantially complementary, hybridize toeach other and not to other nucleic acids that are not substantiallycomplementary to either of the two nucleic acids. For example, specifichybridization includes the hybridization of a primer or capture nucleicacid to a portion of a target nucleic acid (e.g., a template, or adapterportion of a template) that is substantially complementary to the primeror capture nucleic acid. In some embodiments nucleic acids, or portionsthereof, that are configured to specifically hybridize are often about80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% ormore, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more,91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% ormore, 97% or more, 98% or more, 99% or more or 100% complementary toeach other over a contiguous portion of nucleic acid sequence. Aspecific hybridization discriminates over non-specific hybridizationinteractions (e.g., two nucleic acids that a not configured tospecifically hybridize, e.g., two nucleic acids that are 80% or less,70% or less, 60% or less or 50% or less complementary) by about 2-foldor more, often about 10-fold or more, and sometimes about 100-fold ormore, 1000-fold or more, 10,000-fold or more, 100,000-fold or more, or1,000,000-fold or more. Two nucleic acid strands that are hybridized toeach other can form a duplex, which comprises a double stranded portionof nucleic acid.

As used herein, the term “stringent condition” refers to condition(s)under which a polynucleotide probe or primer will hybridizepreferentially to its target sequence, and to a lesser extent to, or notat all to, other sequences. A “stringent hybridization” and “stringenthybridization wash conditions” in the context of nucleic acidhybridization are sequence dependent, and are different under differentenvironmental parameters.

As used herein, the term “nucleic acid” refers to nucleotides (e.g.,deoxyribonucleotides or ribonucleotides) and polymers thereof in eithersingle-, double- or multiple-stranded form, or complements thereof. Theterms “polynucleotide,” “oligonucleotide,” “oligo” or the like refer, inthe usual and customary sense, to a sequence of nucleotides. The term“nucleotide” refers, in the usual and customary sense, to a single unitof a polynucleotide, i.e., a monomer. Nucleotides can beribonucleotides, deoxyribonucleotides, or modified versions thereof.Examples of polynucleotides contemplated herein include single anddouble stranded DNA, single and double stranded RNA, and hybridmolecules having mixtures of single and double stranded DNA and RNA withlinear or circular framework. Non-limiting examples of polynucleotidesinclude a gene, a gene fragment, an exon, an intron, intergenic DNA(including, without limitation, heterochromatic DNA), messenger RNA(mRNA), transfer RNA, ribosomal RNA, a ribozyme, cDNA, a recombinantpolynucleotide, a branched polynucleotide, a plasmid, a vector, isolatedDNA of a sequence, isolated RNA of a sequence, a nucleic acid probe, anda primer. Polynucleotides useful in the methods of the disclosure maycomprise natural nucleic acid sequences and variants thereof, artificialnucleic acid sequences, or a combination of such sequences.

A polynucleotide is typically composed of a specific sequence of fournucleotide bases: adenine (A); cytosine (C); guanine (G); and thymine(T) (uracil (U) for thymine (T) when the polynucleotide is RNA). Thus,the term “polynucleotide sequence” is the alphabetical representation ofa polynucleotide molecule; alternatively, the term may be applied to thepolynucleotide molecule itself. This alphabetical representation can beinput into databases in a computer having a central processing unit andused for bioinformatics applications such as functional genomics andhomology searching. Polynucleotides may optionally include one or morenon-standard nucleotide(s), nucleotide analog(s) and/or modifiednucleotides.

As used herein, the term “polynucleotide template” or “template nucleicacid” refers to any polynucleotide molecule that may be bound by apolymerase and utilized as a template for nucleic acid synthesis. Asused herein, the term “polynucleotide primer” refers to anypolynucleotide molecule that may hybridize to a polynucleotide template,be bound by a polymerase, and be extended in a template-directed processfor nucleic acid synthesis, such as in a PCR or sequencing reaction.Polynucleotide primers attached to a core polymer within a core arereferred to as “core polynucleotide primers.” A primer can be of anylength depending on the particular technique it will be used for. Forexample, PCR primers are generally between 10 and 40 nucleotides inlength. The length and complexity of the nucleic acid fixed onto thenucleic acid template may vary. One of skill can adjust these factors toprovide optimum hybridization and signal production for a givenhybridization procedure. The primer permits the addition of a nucleotideresidue thereto, or oligonucleotide or polynucleotide synthesistherefrom, under suitable conditions. In an embodiment the primer is aDNA primer, i.e., a primer consisting of, or largely consisting of,deoxyribonucleotide residues. The primers are designed to have asequence that is the complement of a region of template/target DNA towhich the primer hybridizes. The addition of a nucleotide residue to the3′ end of a primer by formation of a phosphodiester bond results in aDNA extension product. The addition of a nucleotide residue to the 3′end of the DNA extension product by formation of a phosphodiester bondresults in a further DNA extension product. In another embodiment theprimer is an RNA primer. In embodiments, a primer is hybridized to atarget polynucleotide.

In general, the term “target polynucleotide” refers to a nucleic acidmolecule or polynucleotide in a starting population of nucleic acidmolecules having a target sequence whose presence, amount, and/ornucleotide sequence, or changes in one or more of these, are desired tobe determined. In general, the term “target sequence” refers to anucleic acid sequence on a single strand of nucleic acid. The targetsequence may be a portion of a gene, a regulatory sequence, genomic DNA,cDNA, RNA including mRNA, miRNA, rRNA, or others. The target sequencemay be a target sequence from a sample or a secondary target such as aproduct of an amplification reaction. A target polynucleotide is notnecessarily any single molecule or sequence. For example, a targetpolynucleotide may be any one of a plurality of target polynucleotidesin a reaction, or all polynucleotides in a given reaction, depending onthe reaction conditions. For example, in a nucleic acid amplificationreaction with random primers, all polynucleotides in a reaction may beamplified. As a further example, a collection of targets may besimultaneously assayed using polynucleotide primers directed to aplurality of targets in a single reaction. As yet another example, allor a subset of polynucleotides in a sample may be modified by theaddition of a primer-binding sequence (such as by the ligation ofadapters containing the primer binding sequence), rendering eachmodified polynucleotide a target polynucleotide in a reaction with thecorresponding primer polynucleotide(s).

As used herein, the term “analogue”, in reference to a chemicalcompound, refers to a compound having a structure similar to that ofanother one, but differing from it in respect of one or more differentatoms, functional groups, or substructures that are replaced with one ormore other atoms, functional groups, or substructures. In the context ofa nucleotide useful in practicing the invention, a nucleotide analogrefers to a compound that, like the nucleotide of which it is an analog,can be incorporated into a nucleic acid molecule (e.g., an extensionproduct) by a suitable polymerase, for example, a DNA polymerase in thecontext of a dNTP analogue. The terms also encompass nucleic acidscontaining known nucleotide analogs or modified backbone residues orlinkages, which are synthetic, naturally occurring, and non-naturallyoccurring, which have similar binding properties as the referencenucleic acid, and which are metabolized in a manner similar to thereference nucleotides. Examples of such analogs include, withoutlimitation, phosphodiester derivatives including, e.g., phosphoramidate,phosphorodiamidate, phosphorothioate (also known as phosphothioatehaving double bonded sulfur replacing oxygen in the phosphate),phosphorodithioate, phosphonocarboxylic acids, phosphonocarboxylates,phosphonoacetic acid, phosphonoformic acid, methyl phosphonate, boronphosphonate, or O-methylphosphoroamidite linkages (see, e.g., seeEckstein, OLIGONUCLEOTIDES AND ANALOGUES: A PRACTICAL APPROACH, OxfordUniversity Press) as well as modifications to the nucleotide bases suchas in 5-methyl cytidine or pseudouridine; and peptide nucleic acidbackbones and linkages. Other analog nucleic acids include those withpositive backbones; non-ionic backbones, modified sugars, and non-ribosebackbones (e.g., phosphorodiamidate morpholino oligos or locked nucleicacids (LNA) as known in the art), including those described in U.S. Pat.Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series580, CARBOHYDRATE MODIFICATIONS IN ANTISENSE RESEARCH, Sanghui & Cook,eds. Nucleic acids containing one or more carbocyclic sugars are alsoincluded within one definition of nucleic acids. Modifications of theribose-phosphate backbone may be done for a variety of reasons, e.g., toincrease the stability and half-life of such molecules in physiologicalenvironments or as primers attached to a polymer. Mixtures of naturallyoccurring nucleic acids and analogs can be made; alternatively, mixturesof different nucleic acid analogs, and mixtures of naturally occurringnucleic acids and analogs may be made. In embodiments, theinternucleotide linkages in DNA are phosphodiester, phosphodiesterderivatives, or a combination of both.

As used herein, the term “modified nucleotide” refers to nucleotidemodified in some manner. Typically, a nucleotide contains a single5-carbon sugar moiety, a single nitrogenous base moiety and 1 to threephosphate moieties. In embodiments, a nucleotide can include a blockingmoiety and/or a label moiety. A blocking moiety on a nucleotide preventsformation of a covalent bond between the 3′ hydroxyl moiety of thenucleotide and the 5′ phosphate of another nucleotide. A blocking moietyon a nucleotide can be reversible, whereby the blocking moiety can beremoved or modified to allow the 3′ hydroxyl to form a covalent bondwith the 5′ phosphate of another nucleotide. A blocking moiety can beeffectively irreversible under particular conditions used in a methodset forth herein. In embodiments, the blocking moiety is attached to the3′ oxygen of the nucleotide and is independently —NH₂, —CN, —CH₃, C₂-C₆allyl (e.g., —CH₂—CH═CH₂), methoxyalkyl (e.g., —CH₂—O—CH₃), or —CH₂N₃.In embodiments, the blocking moiety is attached to the 3′ oxygen of thenucleotide and is independently

A label moiety of a nucleotide can be any moiety that allows thenucleotide to be detected, for example, using a spectroscopic method.Exemplary label moieties are fluorescent labels, mass labels,chemiluminescent labels, electrochemical labels, detectable labels andthe like. One or more of the above moieties can be absent from anucleotide used in the methods and compositions set forth herein. Forexample, a nucleotide can lack a label moiety or a blocking moiety orboth. Examples of nucleotide analogs include, without limitation,7-deaza-adenine, 7-deaza-guanine, the analogs of deoxynucleotides shownherein, analogs in which a label is attached through a cleavable linkerto the 5-position of cytosine or thymine or to the 7-position ofdeaza-adenine or deaza-guanine, and analogs in which a small chemicalmoiety is used to cap the —OH group at the 3′-position of deoxyribose.As used herein, the terms “blocking moiety,” “reversible blockinggroup,” “reversible terminator,” and “reversible terminator moiety” areused in accordance with their plain and ordinary meanings and refer to acleavable moiety which does not interfere with incorporation of anucleotide comprising it by a polymerase (e.g., DNA polymerase, modifiedDNA polymerase), but prevents further strand extension until removed(“unblocked”). For example, a reversible terminator may refer to ablocking moiety located, for example, at the 3′ position of thenucleotide and may be a chemically cleavable moiety such as an allylgroup, an azidomethyl group or a methoxymethyl group, or may be anenzymatically cleavable group such as a phosphate ester. Suitablenucleotide blocking moieties are described in applications WO2004/018497, U.S. Pat. Nos. 7,057,026, 7,541,444, WO 96/07669, U.S. Pat.Nos. 5,763,594, 5,808,045, 5,872,244 and 6,232,465 the contents of whichare incorporated herein by reference in their entirety. The nucleotidesmay be labelled or unlabeled. They may be modified with reversibleterminators useful in methods provided herein and may be 3-O-blockedreversible or 3-unblocked reversible terminators. In nucleotides with3-O-blocked reversible terminators, the blocking group may berepresented as —OR [reversible terminating (capping) group], wherein Ois the oxygen atom of the 3-OH of the pentose and R is the blockinggroup, while the label is linked to the base, which acts as a reporterand can be cleaved. The 3-O-blocked reversible terminators are known inthe art, and may be, for instance, a 3′-ONH₂ reversible terminator, a3-O-allyl reversible terminator, or a 3-O-azidomethyl reversibleterminator. In embodiments, the reversible terminator moiety is

as described in U.S. Pat. No. 10,738,072, which is incorporated hereinby reference for all purposes. In embodiments, the reversible terminatormoiety is

As used herein, the term “core” refers to a polymer within whichpolynucleotide primers are attached, and that is surrounded by a “shellpolymer” to which no polynucleotide primers are attached. The presenceof the polynucleotide primer within the core permits a nucleic acidamplification reaction to take place, while the shell polymer provides aphysical barrier between amplification reactions in adjacent cores. Thecores are “surrounded” by the shell polymer in the sense that the shellpolymer completely covers each core, and no core is in direct contactwith any other core. The shell layer may enclose (e.g., surround,encapsulate, envelope) a core. In embodiments, each core surrounded bythe shell polymer forms a discrete particle, the outer surface of whichis defined by the shell polymer. In embodiments, the shells of discretecore-shell particles suspended in a container (e.g., a well, tube, orflow cell) expands, to fill any space between adjacent particles. Insuch cases, the boundaries of individual particles may no longer bereadily discernable, but each core remains separated from each other bythe shell polymer surrounding each, which can be readily observed by,e.g., detecting products of a nucleic acid amplification reaction. Thecore polymer may itself surround a solid support particle, such as aglass, ceramic, metal, silica, magnetic, or paramagnetic particle (e.g.,a 500 nm silica nanoparticle). Solid support particles may be composedof any appropriate material. In embodiments, the support particle is anamorphous solid. In embodiments, the support particle is a crystallinesolid. For example, solid support particles may include appropriatemetals and metal oxides thereof (a metal particle core), carbon (anorganic particle core) silica and oxides thereof (a silica particlecore) or boron and oxides thereof (a boron particle core). For example,the core/shell layers may be formed around a supporting bead(alternatively referred to as a support particle), for example, asilica, magnetic, or paramagnetic bead. The term “support particle” asused herein may refer to any particle or substance having a diameter inthe micrometer range, such as a “microparticle,” which typically has adiameter of approximately 1 μm and higher, or a “nanoparticle,” whichtypically has a diameter of 1 nm to 1 μm. The core, optionally includinga solid silica support particle, may be referred to herein as ananoparticle core wherein the longest diameter is less than 1000nanometers. Lengths and sizes of particles and their surrounding coresas described herein may be measured using Transmission ElectronMicroscopy (TEM). The term “silica” is used according to its plain andordinary meaning and refers to a composition (e.g. a solid compositionsuch as a particle) containing oxides of silicon such as Si atoms (e.g.,in a tetrahedral coordination) with 4 oxygen atoms surrounding a centralSi atom. A silica support particle may refer to a particle including amatrix of silicon-oxygen bonds.

As used herein, the term “polymer” refers to macromolecules having oneor more structurally unique repeating units. The repeating units arereferred to as “monomers,” which are polymerized for the polymer.Typically, a polymer is formed by monomers linked in a chain-likestructure. A polymer formed entirely from a single type of monomer isreferred to as a “homopolymer.” A polymer formed from two or more uniquerepeating structural units may be referred to as a “copolymer.” Apolymer may be linear or branched, and may be random, block, polymerbrush, hyperbranched polymer, bottlebrush polymer, dendritic polymer, orpolymer micelles. The term “polymer” includes homopolymers, copolymers,tripolymers, tetra polymers and other polymeric molecules made frommonomeric subunits. Copolymers include alternating copolymers, periodiccopolymers, statistical copolymers, random copolymers, block copolymers,linear copolymers and branched copolymers. The term “polymerizablemonomer” is used in accordance with its meaning in the art of polymerchemistry and refers to a compound that may covalently bind chemicallyto other monomer molecules (such as other polymerizable monomers thatare the same or different) to form a polymer.

Polymers can be hydrophilic, hydrophobic or amphiphilic, as known in theart. Thus, “hydrophilic polymers” are substantially miscible with waterand include, but are not limited to, polyethylene glycol and the like.“Hydrophobic polymers” are substantially immiscible with water andinclude, but are not limited to, polyethylene, polypropylene,polybutadiene, polystyrene, polymers disclosed herein, and the like.“Amphiphilic polymers” have both hydrophilic and hydrophobic propertiesand are typically copolymers having hydrophilic segment(s) andhydrophobic segment(s). Polymers include homopolymers, randomcopolymers, and block copolymers, as known in the art. The term“homopolymer” refers, in the usual and customary sense, to a polymerhaving a single monomeric unit. The term “copolymer” refers to a polymerderived from two or more monomeric species. The term “random copolymer”refers to a polymer derived from two or more monomeric species with nopreferred ordering of the monomeric species. The term “block copolymer”refers to polymers having two or homopolymer subunits linked by covalentbond. Thus, the term “hydrophobic homopolymer” refers to a homopolymerwhich is hydrophobic. The term “hydrophobic block copolymer” refers totwo or more homopolymer subunits linked by covalent bonds and which ishydrophobic.

As used herein, the term “hydrogel” refers to a three-dimensionalpolymeric structure that is substantially insoluble in water, but whichis capable of absorbing and retaining large quantities of water to forma substantially stable, often soft and pliable, structure. Inembodiments, water can penetrate in between polymer chains of a polymernetwork, subsequently causing swelling and the formation of a hydrogel.In embodiments, hydrogels are super-absorbent (e.g., containing morethan about 90% water) and can be comprised of natural or syntheticpolymers. In some embodiments, the hydrogel polymer includes 60-90%fluid, such as water, and 10-30% polymer. In certain embodiments, thewater content of hydrogel is about 70-80%.

Hydrogels may be prepared by cross-linking hydrophilic biopolymers orsynthetic polymers. Thus, in some embodiments, the hydrogel may includea crosslinker. As used herein, the term “crosslinker” refers to amolecule that can form a three-dimensional network when reacted with theappropriate base monomers. Examples of the hydrogel polymers, which mayinclude one or more crosslinkers, include but are not limited to,hyaluronans, chitosans, agar, heparin, sulfate, cellulose, alginates(including alginate sulfate), collagen, dextrans (including dextransulfate), pectin, carrageenan, polylysine, gelatins (including gelatintype A), agarose,(meth)acrylate-oligolactide-PEO-oligolactide-(meth)acrylate, PEO-PPO-PEOcopolymers (Pluronics), poly(phosphazene), poly(methacrylates),poly(N-vinylpyrrolidone), PL(G)A-PEO-PL(G)A copolymers, poly(ethyleneimine), polyethylene glycol (PEG)-thiol, PEG-acrylate, acrylamide,N,N′-bis(acryloyl)cystamine, PEG, polypropylene oxide (PPO), polyacrylicacid, poly(hydroxyethyl methacrylate) (PHEMA), poly(methyl methacrylate)(PMMA), poly(N-isopropylacrylamide) (PNIPAAm), poly(lactic acid) (PLA),poly(lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL),poly(vinylsulfonic acid) (PVSA), poly(L-aspartic acid), poly(L-glutamicacid), bisacrylamide, diacrylate, diallylamine, triallylamine, divinylsulfone, diethyleneglycol diallyl ether, ethyleneglycol diacrylate,polymethyleneglycol diacrylate, polyethyleneglycol diacrylate,trimethylopropoane trimethacrylate, ethoxylated trimethylol triacrylate,or ethoxylated pentaerythritol tetracrylate, or combinations thereof.Thus, for example, a combination may include a polymer and acrosslinker, for example polyethylene glycol (PEG)-thiol/PEG-acrylate,acrylamide/N,N′-bis(acryloyl)cystamine (BACy), or PEG/polypropyleneoxide (PPO).

The term “array” as used herein, refers to a container (e.g., amultiwell container, reaction vessel, or flow cell) including aplurality of features (e.g., wells). For example, an array may include acontainer with a plurality of wells. In embodiments, the array is amicroplate. In embodiments, the array is a flow cell.

The term “microplate,” “microtiter plate,” or “multiwell plate” as usedherein, refers to a substrate comprising a surface, the surfaceincluding a plurality of chambers or wells separated from each other byinterstitial regions on the surface. In embodiments, the microplate hasdimensions as provided and described by American National StandardsInstitute (ANSI) and Society for Laboratory Automation And Screening(SLAS); for example the tolerances and dimensions set forth in ANSI SLAS1-2004 (R2012); ANSI SLAS 2-2004 (R2012); ANSI SLAS 3-2004 (R2012); ANSISLAS 4-2004 (R2012); and ANSI SLAS 6-2012, which are incorporated hereinby reference. The dimensions of the microplate as described herein andthe arrangement of the reaction chambers may be compatible with anestablished format for automated laboratory equipment. In embodiments,the device described herein provides methods for high-throughputscreening. High-throughput screening (HTS) refers to a process that usesa combination of modern robotics, data processing and control software,liquid handling devices, and/or sensitive detectors, to efficientlyprocess a large amount of (e.g., thousands, hundreds of thousands, ormillions) samples in biochemical, genetic, or pharmacologicalexperiments, either in parallel or in sequence, within a reasonablyshort period of time (e.g., days). Preferably, the process is amenableto automation, such as robotic simultaneous handling of 96 samples, 384samples, 1536 samples or more. A typical HTS robot tests up to 100,000to a few hundred thousand compounds per day. The samples are often insmall volumes, such as no more than 1 mL, 500 μl, 200 μl, 100 μl, 50 μlor less. Through this process, one can rapidly identify activecompounds, small molecules, antibodies, proteins, or polynucleotides ina cell.

The reaction chambers may be provided as wells, for example an array ormicroplate may contain 2, 4, 6, 12, 24, 48, 96, 384, or 1536 samplewells. In embodiments, the 96 and 384 wells are arranged in a 2:3rectangular matrix. In embodiments, the 24 wells are arranged in a 3:8rectangular matrix. In embodiments, the 48 wells are arranged in a 3:4rectangular matrix. In embodiments, the reaction chamber is a microscopeslide (e.g., a glass slide about 75 mm by about 25 mm). In embodiments,the slide is a concavity slide (e.g., the slide includes a depression).In embodiments, the slide includes a coating for enhanced cell adhesion(e.g., poly-L-lysine, silanes, carbon nanotubes, polymers, epoxy resins,or gold). In embodiments, the microplate is about 5 inches by about 3.33inches, and includes a plurality of 5 mm diameter wells. In embodiments,the microplate is about 5 inches by about 3.33 inches, and includes aplurality of 6 mm diameter wells. In embodiments, the microplate isabout 5 inches by about 3.33 inches, and includes a plurality of 7 mmdiameter wells. In embodiments, the microplate is about 5 inches byabout 3.33 inches, and includes a plurality of 7.5 mm diameter wells. Inembodiments, the microplate is 5 inches by 3.33 inches, and includes aplurality of 7.5 mm diameter wells. In embodiments, the microplate isabout 5 inches by about 3.33 inches, and includes a plurality of 8 mmdiameter wells. In embodiments, the microplate is a flat glass orplastic tray in which an array of wells are formed, wherein each wellcan hold between from a few microliters to hundreds of microliters offluid reagents and samples.

The terms “iniferter mediated polymerization” and the like refer, in theusual and customary sense, to polymerization employing an “iniferter”which, as known in the art, is a chemical compound that simultaneouslyacts as initiator, transfer agent, and terminator in controlled freeradical polymerization reactions, e.g., dithiocarbamates. See, e.g.,Otsu, T., & Yashida, M., Mackromol. Chem., Rapid Commun., 1982,3:127-132.

The terms “stable free radical mediated polymerization,” “SRFP,” and thelike refer, in the usual and customary sense, to polymerizationreactions wherein the coupling of the stable free radical with thepolymeric radical is sufficiently reversible that the termination stepis reversible, and the propagating radical concentration can be limitedto levels that allow for controlled polymerization. See e.g., Veregin,R. P. N., et al., Macromolecules 1993, 26:5316-5320.

The terms “atom transfer radical polymerization,” “ATRP” and the likerefer, in the usual and customary sense, to methods of polymerizationemploying a transition metal catalyst, wherein the atom transfer step isthe key step in the reaction responsible for uniform polymer chaingrowth. See e.g., Kato, M., et al., Macromolecules 1995, 28:1721-1723;Wang, J. & Matyjaszewski, K., J. Am. Chem. Soc. 1995, 117:5614-5615.

The terms “reversible addition fragmentation chain transferpolymerization,” “RAFT” and the like refer, in the usual and customarysense, to methods of polymerization which use a chain transfer agent inthe form of a thiocarbonylthio compound or the like to afford controlover the generated molecular weight and polydispersity during afree-radical polymerization. See e.g., Yeole, N., Synlett. 2010(10):1572-1573; Moad, G., et al., Aust. J Chem., 2005, 58:379-410.

As used herein, the term “discrete particles” refers to physicallydistinct particles having discernible boundaries. The term “particle”does not indicate any particular shape. The shapes and sizes of acollection of particles may be different or about the same (e.g., withina desired range of dimensions, or having a desired average or minimumdimension). A particle may be substantially spherical (e.g.,microspheres) or have a non-spherical or irregular shape, such as cubic,cuboid, pyramidal, cylindrical, conical, oblong, or disc-shaped, and thelike. In embodiments, the particle has the shape of a sphere, cylinder,spherocylinder, or ellipsoid. Discrete particles collected in acontainer and contacting one another will define a bulk volumecontaining the particles, and will typically leave some internalfraction of that bulk volume unoccupied by the particles, even whenpacked closely together.

In embodiments, cores and/or core-shell particles are approximatelyspherical. As used herein the term “spherical” refers to structureswhich appear substantially or generally of spherical shape to the humaneye, and does not require a sphere to a mathematical standard. In otherwords, “spherical” cores or particles are generally spheroidal in thesense of resembling or approximating to a sphere. In embodiments, thediameter of a spherical core or particle is substantially uniform, e.g.,about the same at any point, but may contain imperfections, such asdeviations of up to 1, 2, 3, 4, 5 or up to 10%. Because cores orparticles may deviate from a perfect sphere, the term “diameter” refersto the longest dimension of a given core or particle. Likewise, polymershells are not necessarily of perfect uniform thickness all around agiven core. Thus, the term “thickness” in relation to a polymerstructure (e.g., a shell polymer of a core-shell particle) refers to theaverage thickness of the polymer layer.

As used herein, the term “channel” refers to a passage in or on asubstrate material that directs the flow of a fluid. A channel may runalong the surface of a substrate, or may run through the substratebetween openings in the substrate. A channel can have a cross sectionthat is partially or fully surrounded by substrate material (e.g., afluid impermeable substrate material). For example, a partiallysurrounded cross section can be a groove, trough, furrow or gutter thatinhibits lateral flow of a fluid. The transverse cross section of anopen channel can be, for example, U-shaped, V-shaped, curved, angular,polygonal, or hyperbolic. A channel can have a fully surrounded crosssection such as a tunnel, tube, or pipe. A fully surrounded channel canhave a rounded, circular, elliptical, square, rectangular, or polygonalcross section. In particular embodiments, a channel can be located in aflow cell, for example, being embedded within the flow cell. A channelin a flow cell can include one or more windows that are transparent tolight in a particular region of the wavelength spectrum. In embodiments,the channel contains one or more polymers of the disclosure. Inembodiments, the channel is filled by the one or more polymers, and flowthrough the channel (e.g., as in a sample fluid) is directed through thepolymer in the channel. In embodiments, the assay is in a channel of aflow cell.

As used herein, the term “substrate” refers to a solid support material.The substrate can be non-porous or porous. The substrate can be rigid orflexible. As used herein, the terms “solid support” and “solid surface”refers to discrete solid or semi-solid surface. A solid support mayencompass any type of solid, porous, or hollow sphere, ball, cylinder,or other similar configuration composed of plastic, ceramic, metal, orpolymeric material (e.g., hydrogel) onto which a nucleic acid may beimmobilized (e.g., covalently or non-covalently). A nonporous substrategenerally provides a seal against bulk flow of liquids or gases.Exemplary solid supports include, but are not limited to, glass andmodified or functionalized glass, plastics (including acrylics,polystyrene and copolymers of styrene and other materials,polypropylene, polyethylene, polybutylene, polyurethanes, Teflon™,cyclic olefin copolymers, polyimides etc.), nylon, ceramics, resins,Zeonor, silica or silica-based materials including silicon and modifiedsilicon, carbon, metals, inorganic glasses, optical fiber bundles,photopatternable dry film resists, UV-cured adhesives and polymers.Particularly useful solid supports for some embodiments have at leastone surface located within a flow cell. Solid surfaces can also bevaried in their shape depending on the application in a method describedherein. For example, a solid surface useful herein can be planar, orcontain regions which are concave or convex. In embodiments, thegeometry of the concave or convex regions (e.g., wells) of the solidsurface conform to the size and shape of the particle (e.g., see FIG.1C) to maximize the contact between as substantially circular particle.In embodiments, the wells of an array are randomly located such thatnearest neighbor features have random spacing between each other.Alternatively, in embodiments the spacing between the wells can beordered, for example, forming a regular pattern. The term solidsubstrate is encompassing of a substrate (e.g., a flow cell) having asurface comprising a polymer coating covalently attached thereto. Inembodiments, the solid substrate is a flow cell. The term “flow cell” asused herein refers to a chamber including a solid surface across whichone or more fluid reagents can be flowed. Examples of flow cells andrelated fluidic systems and detection platforms that can be readily usedin the methods of the present disclosure are described, for example, inBentley et al., Nature 456:53-59 (2008). In embodiments, a substratecomprises a surface (e.g., a surface of a flow cell, a surface of atube, a surface of a chip), for example a metal surface (e.g., steel,gold, silver, aluminum, silicon and copper). In embodiments, a substrate(e.g., a substrate surface) is coated and/or comprises functional groupsand/or inert materials. In certain embodiments a substrate comprises abead, a chip, a capillary, a plate, a membrane, a wafer (e.g., siliconwafers), a comb, or a pin for example. In embodiments, a substratecomprises a bead and/or a nanoparticle. A substrate can be made of asuitable material, non-limiting examples of which include a plastic or asuitable polymer (e.g., polycarbonate, poly(vinyl alcohol),poly(divinylbenzene), polystyrene, polyamide, polyester, polyvinylidenedifluoride (PVDF), polyethylene, polyurethane, polypropylene, and thelike), borosilicate, glass, nylon, Wang resin, Merrifield resin, metal(e.g., iron, a metal alloy, sepharose, agarose, polyacrylamide, dextran,cellulose and the like or combinations thereof. In some embodiments asubstrate comprises a magnetic material (e.g., iron, nickel, cobalt,platinum, aluminum, and the like). In embodiments, a substrate comprisesa magnetic bead (e.g., DYNABEADS®, hematite, AMPure XP). Magnets can beused to purify and/or capture nucleic acids bound to certain substrates(e.g., substrates comprising a metal or magnetic material). The flowcell is typically a glass slide containing small fluidic channels (e.g.,a glass slide 75 mm×25 mm×1 mm having one or more channels), throughwhich sequencing solutions (e.g., polymerases, nucleotides, and buffers)may traverse. Though typically glass, suitable flow cell materials mayinclude polymeric materials, plastics, silicon, quartz (fused silica),Borofloat® glass, silica, silica-based materials, carbon, metals, anoptical fiber or optical fiber bundles, sapphire, or plastic materialssuch as COCs and epoxies. The particular material can be selected basedon properties desired for a particular use. For example, materials thatare transparent to a desired wavelength of radiation are useful foranalytical techniques that will utilize radiation of the desiredwavelength. Conversely, it may be desirable to select a material thatdoes not pass radiation of a certain wavelength (e.g., being opaque,absorptive, or reflective). In embodiments, the material of the flowcell is selected due to the ability to conduct thermal energy. Inembodiments, a flow cell includes inlet and outlet ports and a flowchannel extending there between.

The term “surface” is intended to mean an external part or externallayer of a substrate. The surface can be in contact with anothermaterial such as a gas, liquid, gel, polymer, organic polymer, secondsurface of a similar or different material, metal, or coat. The surface,or regions thereof, can be substantially flat. The substrate and/or thesurface can have surface features such as wells, pits, channels, ridges,raised regions, pegs, posts or the like.

The term “well” refers to a discrete concave feature or depression in asubstrate having a surface opening that is completely surrounded byinterstitial region(s) of the surface. Wells can have any of a varietyof shapes at their opening in a surface including but not limited toround, elliptical, square, polygonal, or star shaped (i.e., star shapedwith any number of vertices). The cross section of a well takenorthogonally with the surface may be curved, square, polygonal,hyperbolic, conical, or angular. The wells of a microplate may beavailable in different shapes, for example F-Bottom: flat bottom;C-Bottom: bottom with minimal rounded edges; V-Bottom: V-shaped bottom;or U-Bottom: U-shaped bottom. In embodiments, the well is substantiallysquare. In embodiments, the well is square. In embodiments, the well isF-bottom. In embodiments, the microplate includes 24 substantially roundflat bottom wells. In embodiments, the microplate includes 48substantially round flat bottom wells. In embodiments, the microplateincludes 96 substantially round flat bottom wells. In embodiments, themicroplate includes 384 substantially square flat bottom wells.

The discrete regions (i.e., features or wells) may have definedlocations in a regular array, which may correspond to a rectilinearpattern, circular pattern, hexagonal pattern, or the like. Inembodiments, the pattern of wells includes concentric circles ofregions, spiral patterns, rectilinear patterns, hexagonal patterns, andthe like. In embodiments, the pattern of wells is arranged in arectilinear or hexagonal pattern A regular array of such regions isadvantageous for detection and data analysis of signals collected fromthe arrays during an analysis. These discrete regions are separated byinterstitial regions. As used herein, the term “interstitial region”refers to an area in a substrate or on a surface that separates otherareas of the substrate or surface. For example, an interstitial regioncan separate one concave feature of an array from another concavefeature of the array. The two regions that are separated from each othercan be discrete, lacking contact with each other. In another example, aninterstitial region can separate a first portion of a feature from asecond portion of a feature. In embodiments, the interstitial region iscontinuous whereas the features are discrete, for example, as is thecase for an array of wells in an otherwise continuous surface. Theseparation provided by an interstitial region can be partial or fullseparation. In embodiments, interstitial regions have a surface materialthat differs from the surface material of the wells (e.g., theinterstitial region contains a photoresist and the surface of the wellis glass). In embodiments, interstitial regions have a surface materialthat is the same as the surface material of the wells (e.g., both thesurface of the interstitial region and the surface of well contain apassivating polymer or copolymer, as depicted in FIGS. 1A-1C). Inembodiments, interstitial regions have a surface material that is thesame as the surface material of the wells (e.g., both the surface of theinterstitial region and the surface of well contain a polymer orcopolymer).

As used herein, the term “feature” refers a point or area in a patternthat can be distinguished from other points or areas according to itsrelative location. An individual feature can include one or morepolynucleotides. For example, a feature can include a single targetnucleic acid molecule having a particular sequence or a feature caninclude several nucleic acid molecules having the same sequence (and/orcomplementary sequence, thereof). Different molecules that are atdifferent features of a pattern can be differentiated from each otheraccording to the locations of the features in the pattern. Non-limitingexamples of features include wells in a substrate, particles (e.g.,beads) in or on a substrate, polymers in or on a substrate, projectionsfrom a substrate, ridges on a substrate, or channels in a substrate.

As used herein, the terms “sequencing”, “sequence determination”, and“determining a nucleotide sequence”, are used in accordance with theirordinary meaning in the art, and refer to determination of partial aswell as full sequence information of the nucleic acid being sequenced,and particular physical processes for generating such sequenceinformation. That is, the term includes sequence comparisons,fingerprinting, and like levels of information about a target nucleicacid, as well as the express identification and ordering of nucleotidesin a target nucleic acid. The term also includes the determination ofthe identification, ordering, and locations of one, two, or three of thefour types of nucleotides within a target nucleic acid. As used herein,the term “sequencing cycle” is used in accordance with its plain andordinary meaning and refers to incorporating one or more nucleotides(e.g., nucleotide analogues) to the 3′ end of a polynucleotide with apolymerase, and detecting one or more labels that identify the one ormore nucleotides incorporated. In embodiments, one nucleotide (e.g., amodified nucleotide) is incorporated per sequencing cycle. Thesequencing may be accomplished by, for example, sequencing by synthesis,pyrosequencing, and the like. In embodiments, a sequencing cycleincludes extending a complementary polynucleotide by incorporating afirst nucleotide using a polymerase, wherein the polynucleotide ishybridized to a template nucleic acid, detecting the first nucleotide,and identifying the first nucleotide. In embodiments, to begin asequencing cycle, one or more differently labeled nucleotides and a DNApolymerase can be introduced. Following nucleotide addition, signalsproduced (e.g., via excitation and emission of a detectable label) canbe detected to determine the identity of the incorporated nucleotide(based on the labels on the nucleotides). Reagents can then be added toremove the 3′ reversible terminator and to remove labels from eachincorporated base. Reagents, enzymes, and other substances can beremoved between steps by washing. Cycles may include repeating thesesteps, and the sequence of each cluster is read over the multiplerepetitions.

As used herein, the term “extension” or “elongation” is used inaccordance with its plain and ordinary meanings and refer to synthesisby a polymerase of a new polynucleotide strand complementary to atemplate strand by adding free nucleotides (e.g., dNTPs) from a reactionmixture that are complementary to the template in the 5′-to-3′direction. Extension includes condensing the 5′-phosphate group of thedNTPs with the 3′-hydroxy group at the end of the nascent (elongating)polynucleotide strand.

As used herein, the term “sequencing read” is used in accordance withits plain and ordinary meaning and refers to an inferred sequence ofnucleotide bases (or nucleotide base probabilities) corresponding to allor part of a single polynucleotide fragment. A sequencing read mayinclude 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, or morenucleotide bases. In embodiments, a sequencing read includes reading abarcode sequence and a template nucleotide sequence. In embodiments, asequencing read includes reading a template nucleotide sequence. Inembodiments, a sequencing read includes reading a barcode and not atemplate nucleotide sequence. Reads of length 20-40 base pairs (bp) arereferred to as ultra-short. Typical sequencers produce read lengths inthe range of 100-500 bp. Read length is a factor which can affect theresults of biological studies. For example, longer read lengths improvethe resolution of de novo genome assembly and detection of structuralvariants. In embodiments, a sequencing read includes reading a barcodeand a template nucleotide sequence. In embodiments, a sequencing readincludes reading a template nucleotide sequence. In embodiments, asequencing read includes reading a barcode and not a template nucleotidesequence. In embodiments, a sequencing read includes a computationallyderived string corresponding to the detected label. In some embodiments,a sequencing read may include 500, 600, 700, 800, 900, 1,000, 1,100,1,200, 1,300, 1,400, 1,500, or more nucleotide bases.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly indicates otherwise, between the upper and lowerlimit of that range, and any other stated or unstated intervening valuein, or smaller range of values within, that stated range is encompassedwithin the invention. The upper and lower limits of any such smallerrange (within a more broadly recited range) may independently beincluded in the smaller ranges, or as particular values themselves, andare also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

The abbreviations used herein have their conventional meaning within thechemical and biological arts. The chemical structures and formulae setforth herein are constructed according to the standard rules of chemicalvalency known in the chemical arts.

Where substituent groups are specified by their conventional chemicalformulae, written from left to right, they equally encompass thechemically identical substituents that would result from writing thestructure from right to left, e.g., —CH₂O— is equivalent to —OCH₂—.

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a straight (i.e., unbranched) or branchedcarbon chain (or carbon), or combination thereof, which may be fullysaturated, mono- or polyunsaturated and can include mono-, di- andmultivalent radicals. The alkyl may include a designated number ofcarbons (e.g., C₁-C₁₀ means one to ten carbons). In embodiments, thealkyl is fully saturated. In embodiments, the alkyl is monounsaturated.In embodiments, the alkyl is polyunsaturated. Alkyl is an uncyclizedchain. Examples of saturated hydrocarbon radicals include, but are notlimited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl,t-butyl, isobutyl, sec-butyl, methyl, homologs and isomers of, forexample, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. Anunsaturated alkyl group is one having one or more double bonds or triplebonds. Examples of unsaturated alkyl groups include, but are not limitedto, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl),2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl,3-butynyl, and the higher homologs and isomers. An alkoxy is an alkylattached to the remainder of the molecule via an oxygen linker (—O—). Analkyl moiety may be an alkenyl moiety. An alkyl moiety may be an alkynylmoiety. An alkyl moiety may be fully saturated. An alkenyl may includemore than one double bond and/or one or more triple bonds in addition tothe one or more double bonds. An alkynyl may include more than onetriple bond and/or one or more double bonds in addition to the one ormore triple bonds.

The term “alkylene,” by itself or as part of another substituent, means,unless otherwise stated, a divalent radical derived from an alkyl, asexemplified, but not limited by, —CH₂CH₂CH₂CH₂—. Typically, an alkyl (oralkylene) group will have from 1 to 24 carbon atoms, with those groupshaving 10 or fewer carbon atoms being preferred herein. A “lower alkyl”or “lower alkylene” is a shorter chain alkyl or alkylene group,generally having eight or fewer carbon atoms. The term “alkenylene,” byitself or as part of another substituent, means, unless otherwisestated, a divalent radical derived from an alkene. The term “alkynylene”by itself or as part of another substituent, means, unless otherwisestated, a divalent radical derived from an alkyne. In embodiments, thealkylene is fully saturated. In embodiments, the alkylene ismonounsaturated. In embodiments, the alkylene is polyunsaturated. Analkenylene includes one or more double bonds. An alkynylene includes oneor more triple bonds.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chain, orcombinations thereof, including at least one carbon atom and at leastone heteroatom (e.g., O, N, P, Si, and S), and wherein the nitrogen andsulfur atoms may optionally be oxidized, and the nitrogen heteroatom mayoptionally be quaternized. The heteroatom(s) (e.g., N, S, Si, or P) maybe placed at any interior position of the heteroalkyl group or at theposition at which the alkyl group is attached to the remainder of themolecule. Heteroalkyl is an uncyclized chain. Examples include, but arenot limited to: —CH₂—CH₂—O—CH₃, —CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃,—CH₂—S—CH₂—CH₃, —CH₂—S—CH₂, —S(O)—CH₃, —CH₂—CH₂—S(O)₂—CH₃, —CH═CHO—CH₃,—Si(CH₃)₃, —CH₂—CH═N—OCH₃, —CH═CH—N(CH₃)—CH₃, —O—CH₃, —O—CH₂—CH₃, and—CN. Up to two or three heteroatoms may be consecutive, such as, forexample, —CH₂—NH—OCH₃ and —CH₂—O—Si(CH₃)₃. A heteroalkyl moiety mayinclude one heteroatom (e.g., O, N, S, Si, or P). A heteroalkyl moietymay include two optionally different heteroatoms (e.g., O, N, S, Si, orP). A heteroalkyl moiety may include three optionally differentheteroatoms (e.g., O, N, S, Si, or P). A heteroalkyl moiety may includefour optionally different heteroatoms (e.g., O, N, S, Si, or P). Aheteroalkyl moiety may include five optionally different heteroatoms(e.g., O, N, S, Si, or P). A heteroalkyl moiety may include up to 8optionally different heteroatoms (e.g., O, N, S, Si, or P). The term“heteroalkenyl,” by itself or in combination with another term, means,unless otherwise stated, a heteroalkyl including at least one doublebond. A heteroalkenyl may optionally include more than one double bondand/or one or more triple bonds in additional to the one or more doublebonds. The term “heteroalkynyl,” by itself or in combination withanother term, means, unless otherwise stated, a heteroalkyl including atleast one triple bond. A heteroalkynyl may optionally include more thanone triple bond and/or one or more double bonds in additional to the oneor more triple bonds. In embodiments, the heteroalkyl is fullysaturated. In embodiments, the heteroalkyl is monounsaturated. Inembodiments, the heteroalkyl is polyunsaturated.

Similarly, the term “heteroalkylene,” by itself or as part of anothersubstituent, means, unless otherwise stated, a divalent radical derivedfrom heteroalkyl, as exemplified, but not limited by,—CH₂—CH₂—S—CH₂—CH₂— and —CH₂—S—CH₂—CH₂—NH—CH₂—. For heteroalkylenegroups, heteroatoms can also occupy either or both of the chain termini(e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, andthe like). Still further, for alkylene and heteroalkylene linkinggroups, no orientation of the linking group is implied by the directionin which the formula of the linking group is written. For example, theformula —C(O)₂R′— represents both —C(O)₂R′— and —R′C(O)₂—. As describedabove, heteroalkyl groups, as used herein, include those groups that areattached to the remainder of the molecule through a heteroatom, such as—C(O)R′, —C(O)NR′, —NR′R″, —OR′, —SR′, and/or —SO₂R′. Where“heteroalkyl” is recited, followed by recitations of specificheteroalkyl groups, such as —NR′R″ or the like, it will be understoodthat the terms heteroalkyl and —NR′R″ are not redundant or mutuallyexclusive. Rather, the specific heteroalkyl groups are recited to addclarity. Thus, the term “heteroalkyl” should not be interpreted hereinas excluding specific heteroalkyl groups, such as —NR′R″ or the like.The term “heteroalkenylene,” by itself or as part of anothersubstituent, means, unless otherwise stated, a divalent radical derivedfrom a heteroalkene. The term “heteroalkynylene” by itself or as part ofanother substituent, means, unless otherwise stated, a divalent radicalderived from a heteroalkyne. In embodiments, the heteroalkylene is fullysaturated. In embodiments, the heteroalkylene is monounsaturated. Inembodiments, the heteroalkylene is polyunsaturated. A heteroalkenyleneincludes one or more double bonds. A heteroalkynylene includes one ormore triple bonds.

The terms “cycloalkyl” and “heterocycloalkyl,” by themselves or incombination with other terms, mean, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl,” respectively. Cycloalkyl andheterocycloalkyl are not aromatic. Additionally, for heterocycloalkyl, aheteroatom can occupy the position at which the heterocycle is attachedto the remainder of the molecule. Examples of cycloalkyl include, butare not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples ofheterocycloalkyl include, but are not limited to,1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like. A “cycloalkylene” and a“heterocycloalkylene,” alone or as part of another substituent, means adivalent radical derived from a cycloalkyl and heterocycloalkyl,respectively. In embodiments, the cycloalkyl is fully saturated. Inembodiments, the cycloalkyl is monounsaturated. In embodiments, thecycloalkyl is polyunsaturated. In embodiments, the heterocycloalkyl isfully saturated. In embodiments, the heterocycloalkyl ismonounsaturated. In embodiments, the heterocycloalkyl ispolyunsaturated.

In embodiments, the term “cycloalkyl” means a monocyclic, bicyclic, or amulticyclic cycloalkyl ring system. In embodiments, monocyclic ringsystems are cyclic hydrocarbon groups containing from 3 to 8 carbonatoms, where such groups can be saturated or unsaturated, but notaromatic. In embodiments, cycloalkyl groups are fully saturated. Abicyclic or multicyclic cycloalkyl ring system refers to multiple ringsfused together wherein at least one of the fused rings is a cycloalkylring and wherein the multiple rings are attached to the parent molecularmoiety through any carbon atom contained within a cycloalkyl ring of themultiple rings. Examples of monocyclic cycloalkyls include cyclopropyl,cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl,cycloheptyl, and cyclooctyl. Bicyclic cycloalkyl ring systems arebridged monocyclic rings or fused bicyclic rings. In embodiments,bridged monocyclic rings contain a monocyclic cycloalkyl ring where twonon adjacent carbon atoms of the monocyclic ring are linked by analkylene bridge of between one and three additional carbon atoms (i.e.,a bridging group of the form (CH₂)_(w), where w is 1, 2, or 3).Representative examples of bicyclic ring systems include, but are notlimited to, bicyclo[3.1.1]heptane, bicyclo[2.2.1]heptane,bicyclo[2.2.2]octane, bicyclo[3.2.2]nonane, bicyclo[3.3.1]nonane, andbicyclo[4.2.1]nonane. In embodiments, fused bicyclic cycloalkyl ringsystems contain a monocyclic cycloalkyl ring fused to either a phenyl, amonocyclic cycloalkyl, a monocyclic cycloalkenyl, a monocyclicheterocyclyl, or a monocyclic heteroaryl. In embodiments, the bridged orfused bicyclic cycloalkyl is attached to the parent molecular moietythrough any carbon atom contained within the monocyclic cycloalkyl ring.In embodiments, cycloalkyl groups are optionally substituted with one ortwo groups which are independently oxo or thia. In embodiments, thefused bicyclic cycloalkyl is a 5 or 6 membered monocyclic cycloalkylring fused to either a phenyl ring, a 5 or 6 membered monocycliccycloalkyl, a 5 or 6 membered monocyclic cycloalkenyl, a 5 or 6 memberedmonocyclic heterocyclyl, or a 5 or 6 membered monocyclic heteroaryl,wherein the fused bicyclic cycloalkyl is optionally substituted by oneor two groups which are independently oxo or thia. In embodiments,multicyclic cycloalkyl ring systems are a monocyclic cycloalkyl ring(base ring) fused to either (i) one ring system selected from the groupconsisting of a bicyclic aryl, a bicyclic heteroaryl, a bicycliccycloalkyl, a bicyclic cycloalkenyl, and a bicyclic heterocyclyl; or(ii) two other ring systems independently selected from the groupconsisting of a phenyl, a bicyclic aryl, a monocyclic or bicyclicheteroaryl, a monocyclic or bicyclic cycloalkyl, a monocyclic orbicyclic cycloalkenyl, and a monocyclic or bicyclic heterocyclyl. Inembodiments, the multicyclic cycloalkyl is attached to the parentmolecular moiety through any carbon atom contained within the base ring.In embodiments, multicyclic cycloalkyl ring systems are a monocycliccycloalkyl ring (base ring) fused to either (i) one ring system selectedfrom the group consisting of a bicyclic aryl, a bicyclic heteroaryl, abicyclic cycloalkyl, a bicyclic cycloalkenyl, and a bicyclicheterocyclyl; or (ii) two other ring systems independently selected fromthe group consisting of a phenyl, a monocyclic heteroaryl, a monocycliccycloalkyl, a monocyclic cycloalkenyl, and a monocyclic heterocyclyl.Examples of multicyclic cycloalkyl groups include, but are not limitedto tetradecahydrophenanthrenyl, perhydrophenothiazin-1-yl, andperhydrophenoxazin-1-yl.

In embodiments, a cycloalkyl is a cycloalkenyl. The term “cycloalkenyl”is used in accordance with its plain ordinary meaning. In embodiments, acycloalkenyl is a monocyclic, bicyclic, or a multicyclic cycloalkenylring system. A bicyclic or multicyclic cycloalkenyl ring system refersto multiple rings fused together wherein at least one of the fused ringsis a cycloalkenyl ring and wherein the multiple rings are attached tothe parent molecular moiety through any carbon atom contained within acycloalkenyl ring of the multiple rings. In embodiments, monocycliccycloalkenyl ring systems are cyclic hydrocarbon groups containing from3 to 8 carbon atoms, where such groups are unsaturated (i.e., containingat least one annular carbon carbon double bond), but not aromatic.Examples of monocyclic cycloalkenyl ring systems include cyclopentenyland cyclohexenyl. In embodiments, bicyclic cycloalkenyl rings arebridged monocyclic rings or a fused bicyclic rings. In embodiments,bridged monocyclic rings contain a monocyclic cycloalkenyl ring wheretwo non adjacent carbon atoms of the monocyclic ring are linked by analkylene bridge of between one and three additional carbon atoms (i.e.,a bridging group of the form (CH₂)_(w), where w is 1, 2, or 3).Representative examples of bicyclic cycloalkenyls include, but are notlimited to, norbornenyl and bicyclo[2.2.2]oct 2 enyl. In embodiments,fused bicyclic cycloalkenyl ring systems contain a monocycliccycloalkenyl ring fused to either a phenyl, a monocyclic cycloalkyl, amonocyclic cycloalkenyl, a monocyclic heterocyclyl, or a monocyclicheteroaryl. In embodiments, the bridged or fused bicyclic cycloalkenylis attached to the parent molecular moiety through any carbon atomcontained within the monocyclic cycloalkenyl ring. In embodiments,cycloalkenyl groups are optionally substituted with one or two groupswhich are independently oxo or thia. In embodiments, multicycliccycloalkenyl rings contain a monocyclic cycloalkenyl ring (base ring)fused to either (i) one ring system selected from the group consistingof a bicyclic aryl, a bicyclic heteroaryl, a bicyclic cycloalkyl, abicyclic cycloalkenyl, and a bicyclic heterocyclyl; or (ii) two ringsystems independently selected from the group consisting of a phenyl, abicyclic aryl, a monocyclic or bicyclic heteroaryl, a monocyclic orbicyclic cycloalkyl, a monocyclic or bicyclic cycloalkenyl, and amonocyclic or bicyclic heterocyclyl. In embodiments, the multicycliccycloalkenyl is attached to the parent molecular moiety through anycarbon atom contained within the base ring. In embodiments, multicycliccycloalkenyl rings contain a monocyclic cycloalkenyl ring (base ring)fused to either (i) one ring system selected from the group consistingof a bicyclic aryl, a bicyclic heteroaryl, a bicyclic cycloalkyl, abicyclic cycloalkenyl, and a bicyclic heterocyclyl; or (ii) two ringsystems independently selected from the group consisting of a phenyl, amonocyclic heteroaryl, a monocyclic cycloalkyl, a monocycliccycloalkenyl, and a monocyclic heterocyclyl.

In embodiments, the term “heterocycloalkyl” means a monocyclic,bicyclic, or a multicyclic heterocycloalkyl ring system. In embodiments,heterocycloalkyl groups are fully saturated. A bicyclic or multicyclicheterocycloalkyl ring system refers to multiple rings fused togetherwherein at least one of the fused rings is a heterocycloalkyl ring andwherein the multiple rings are attached to the parent molecular moietythrough any atom contained within a heterocycloalkyl ring of themultiple rings. In embodiments, a heterocycloalkyl is a heterocyclyl.The term “heterocyclyl” as used herein, means a monocyclic, bicyclic, ormulticyclic heterocycle. The heterocyclyl monocyclic heterocycle is a 3,4, 5, 6 or 7 membered ring containing at least one heteroatomindependently selected from the group consisting of O, N, and S wherethe ring is saturated or unsaturated, but not aromatic. The 3 or 4membered ring contains 1 heteroatom selected from the group consistingof O, N and S. The 5 membered ring can contain zero or one double bondand one, two or three heteroatoms selected from the group consisting ofO, N and S. The 6 or 7 membered ring contains zero, one or two doublebonds and one, two or three heteroatoms selected from the groupconsisting of O, N and S. The heterocyclyl monocyclic heterocycle isconnected to the parent molecular moiety through any carbon atom or anynitrogen atom contained within the heterocyclyl monocyclic heterocycle.Representative examples of heterocyclyl monocyclic heterocycles include,but are not limited to, azetidinyl, azepanyl, aziridinyl, diazepanyl,1,3-dioxanyl, 1,3-dioxolanyl, 1,3-dithiolanyl, 1,3-dithianyl,imidazolinyl, imidazolidinyl, isothiazolinyl, isothiazolidinyl,isoxazolinyl, isoxazolidinyl, morpholinyl, oxadiazolinyl,oxadiazolidinyl, oxazolinyl, oxazolidinyl, piperazinyl, piperidinyl,pyranyl, pyrazolinyl, pyrazolidinyl, pyrrolinyl, pyrrolidinyl,tetrahydrofuranyl, tetrahydrothienyl, thiadiazolinyl, thiadiazolidinyl,thiazolinyl, thiazolidinyl, thiomorpholinyl, 1,1-dioxidothiomorpholinyl(thiomorpholine sulfone), thiopyranyl, and trithianyl. The heterocyclylbicyclic heterocycle is a monocyclic heterocycle fused to either aphenyl, a monocyclic cycloalkyl, a monocyclic cycloalkenyl, a monocyclicheterocycle, or a monocyclic heteroaryl. The heterocyclyl bicyclicheterocycle is connected to the parent molecular moiety through anycarbon atom or any nitrogen atom contained within the monocyclicheterocycle portion of the bicyclic ring system. Representative examplesof bicyclic heterocyclyls include, but are not limited to,2,3-dihydrobenzofuran-2-yl, 2,3-dihydrobenzofuran-3-yl, indolin-1-yl,indolin-2-yl, indolin-3-yl, 2,3-dihydrobenzothien-2-yl,decahydroquinolinyl, decahydroisoquinolinyl, octahydro-1H-indolyl, andoctahydrobenzofuranyl. In embodiments, heterocyclyl groups areoptionally substituted with one or two groups which are independentlyoxo or thia. In certain embodiments, the bicyclic heterocyclyl is a 5 or6 membered monocyclic heterocyclyl ring fused to a phenyl ring, a 5 or 6membered monocyclic cycloalkyl, a 5 or 6 membered monocycliccycloalkenyl, a 5 or 6 membered monocyclic heterocyclyl, or a 5 or 6membered monocyclic heteroaryl, wherein the bicyclic heterocyclyl isoptionally substituted by one or two groups which are independently oxoor thia. Multicyclic heterocyclyl ring systems are a monocyclicheterocyclyl ring (base ring) fused to either (i) one ring systemselected from the group consisting of a bicyclic aryl, a bicyclicheteroaryl, a bicyclic cycloalkyl, a bicyclic cycloalkenyl, and abicyclic heterocyclyl; or (ii) two other ring systems independentlyselected from the group consisting of a phenyl, a bicyclic aryl, amonocyclic or bicyclic heteroaryl, a monocyclic or bicyclic cycloalkyl,a monocyclic or bicyclic cycloalkenyl, and a monocyclic or bicyclicheterocyclyl. The multicyclic heterocyclyl is attached to the parentmolecular moiety through any carbon atom or nitrogen atom containedwithin the base ring. In embodiments, multicyclic heterocyclyl ringsystems are a monocyclic heterocyclyl ring (base ring) fused to either(i) one ring system selected from the group consisting of a bicyclicaryl, a bicyclic heteroaryl, a bicyclic cycloalkyl, a bicycliccycloalkenyl, and a bicyclic heterocyclyl; or (ii) two other ringsystems independently selected from the group consisting of a phenyl, amonocyclic heteroaryl, a monocyclic cycloalkyl, a monocycliccycloalkenyl, and a monocyclic heterocyclyl. Examples of multicyclicheterocyclyl groups include, but are not limited to10H-phenothiazin-10-yl, 9,10-dihydroacridin-9-yl,9,10-dihydroacridin-10-yl, 10H-phenoxazin-10-yl,10,11-dihydro-5H-dibenzo[b,f]azepin-5-yl,1,2,3,4-tetrahydropyrido[4,3-g]isoquinolin-2-yl,12H-benzo[b]phenoxazin-12-yl, and dodecahydro-1H-carbazol-9-yl.

The terms “halo” or “halogen,” by themselves or as part of anothersubstituent, mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom. Additionally, terms such as “haloalkyl” aremeant to include monohaloalkyl and polyhaloalkyl. For example, the term“halo(C₁-C₄)alkyl” includes, but is not limited to, fluoromethyl,difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl,3-bromopropyl, and the like.

The term “acyl” means, unless otherwise stated, —C(O)R where R is asubstituted or unsubstituted alkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl.

The term “aryl” means, unless otherwise stated, a polyunsaturated,aromatic, hydrocarbon substituent, which can be a single ring ormultiple rings (preferably from 1 to 3 rings) that are fused together(i.e., a fused ring aryl) or linked covalently. A fused ring aryl refersto multiple rings fused together wherein at least one of the fused ringsis an aryl ring. In embodiments, a fused ring aryl refers to multiplerings fused together wherein at least one of the fused rings is an arylring and wherein the multiple rings are attached to the parent molecularmoiety through any carbon atom contained within an aryl ring of themultiple rings. The term “heteroaryl” refers to aryl groups (or rings)that contain at least one heteroatom such as N, O, or S, wherein thenitrogen and sulfur atoms are optionally oxidized, and the nitrogenatom(s) are optionally quaternized. Thus, the term “heteroaryl” includesfused ring heteroaryl groups (i.e., multiple rings fused togetherwherein at least one of the fused rings is a heteroaromatic ring). Inembodiments, the term “heteroaryl” includes fused ring heteroaryl groups(i.e., multiple rings fused together wherein at least one of the fusedrings is a heteroaromatic ring and wherein the multiple rings areattached to the parent molecular moiety through any atom containedwithin a heteroaromatic ring of the multiple rings). A 5,6-fused ringheteroarylene refers to two rings fused together, wherein one ring has 5members and the other ring has 6 members, and wherein at least one ringis a heteroaryl ring. Likewise, a 6,6-fused ring heteroarylene refers totwo rings fused together, wherein one ring has 6 members and the otherring has 6 members, and wherein at least one ring is a heteroaryl ring.And a 6,5-fused ring heteroarylene refers to two rings fused together,wherein one ring has 6 members and the other ring has 5 members, andwherein at least one ring is a heteroaryl ring. A heteroaryl group canbe attached to the remainder of the molecule through a carbon orheteroatom. Non-limiting examples of aryl and heteroaryl groups includephenyl, naphthyl, pyrrolyl, pyrazolyl, pyridazinyl, triazinyl,pyrimidinyl, imidazolyl, pyrazinyl, purinyl, oxazolyl, isoxazolyl,thiazolyl, furyl, thienyl, pyridyl, pyrimidyl, benzothiazolyl,benzoxazoyl benzimidazolyl, benzofuran, isobenzofuranyl, indolyl,isoindolyl, benzothiophenyl, isoquinolyl, quinoxalinyl, quinolyl,1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl,3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl,4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl,2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl,4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl,1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl,3-quinolyl, and 6-quinolyl. Substituents for each of the above notedaryl and heteroaryl ring systems are selected from the group ofacceptable substituents described below. An “arylene” and a“heteroarylene,” alone or as part of another substituent, mean adivalent radical derived from an aryl and heteroaryl, respectively. Aheteroaryl group substituent may be —O— bonded to a ring heteroatomnitrogen.

Spirocyclic rings are two or more rings wherein adjacent rings areattached through a single atom. The individual rings within spirocyclicrings may be identical or different. Individual rings in spirocyclicrings may be substituted or unsubstituted and may have differentsubstituents from other individual rings within a set of spirocyclicrings. Possible substituents for individual rings within spirocyclicrings are the possible substituents for the same ring when not part ofspirocyclic rings (e.g., substituents for cycloalkyl or heterocycloalkylrings). Spirocylic rings may be substituted or unsubstituted cycloalkyl,substituted or unsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkyl or substituted or unsubstituted heterocycloalkylene andindividual rings within a spirocyclic ring group may be any of theimmediately previous list, including having all rings of one type (e.g.,all rings being substituted heterocycloalkylene wherein each ring may bethe same or different substituted heterocycloalkylene). When referringto a spirocyclic ring system, heterocyclic spirocyclic rings means aspirocyclic rings wherein at least one ring is a heterocyclic ring andwherein each ring may be a different ring. When referring to aspirocyclic ring system, substituted spirocyclic rings means that atleast one ring is substituted and each substituent may optionally bedifferent.

The symbol “

” denotes the point of attachment of a chemical moiety to the remainderof a molecule or chemical formula.

The term “oxo,” as used herein, means an oxygen that is double bonded toa carbon atom.

The term “alkylarylene” as an arylene moiety covalently bonded to analkylene moiety (also referred to herein as an alkylene linker). Inembodiments, the alkylarylene group has the formula:

An alkylarylene moiety may be substituted (e.g., with a substituentgroup) on the alkylene moiety or the arylene linker (e.g., at carbons 2,3, 4, or 6) with halogen, oxo, —N₃, —CF₃, —CCl₃, —CBr₃, —CI₃, —CN, —CHO,—OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₂CH₃, —SO₃H, —OSO₃H, —SO₂NH₂,—NHNH₂, —ONH₂, —NHC(O)NHNH₂, substituted or unsubstituted C₁-C₅ alkyl orsubstituted or unsubstituted 2 to 5 membered heteroalkyl). Inembodiments, the alkylarylene is unsubstituted.

Each of the above terms (e.g., “alkyl,” “heteroalkyl,” “cycloalkyl,”“heterocycloalkyl,” “aryl,” and “heteroaryl”) includes both substitutedand unsubstituted forms of the indicated radical. Preferred substituentsfor each type of radical are provided below.

Substituents for the alkyl and heteroalkyl radicals (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) can be one or more of a variety of groups selectedfrom, but not limited to, —OR′, ═O, ═NR′, ═N—OR′, —NR′R″, —SR′, halogen,—SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —NR′NR″R′″,—ONR′R″, —NR′C(O)NR″NR′″R″ ″, —CN, —NO₂, —NR′SO₂R″, —NR′C(O)R″,—NR′C(O)—OR″, —NR′OR″, in a number ranging from zero to (2m′+1), wherem′ is the total number of carbon atoms in such radical. R, R′, R″, R′″,and R″ ″ each preferably independently refer to hydrogen, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl (e.g., aryl substituted with 1-3 halogens),substituted or unsubstituted heteroaryl, substituted or unsubstitutedalkyl, alkoxy, or thioalkoxy groups, or arylalkyl groups. When acompound described herein includes more than one R group, for example,each of the R groups is independently selected as are each R′, R″, R′″,and R″″ group when more than one of these groups is present. When R′ andR″ are attached to the same nitrogen atom, they can be combined with thenitrogen atom to form a 4-, 5-, 6-, or 7-membered ring. For example,—NR′R″ includes, but is not limited to, 1-pyrrolidinyl and4-morpholinyl. From the above discussion of substituents, one of skillin the art will understand that the term “alkyl” is meant to includegroups including carbon atoms bound to groups other than hydrogengroups, such as haloalkyl (e.g., —CF₃ and —CH₂CF₃) and acyl (e.g.,—C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and the like).

Similar to the substituents described for the alkyl radical,substituents for the aryl and heteroaryl groups are varied and areselected from, for example: —OR′, —NR′R″, —SR′, halogen, —SiR′R″R′″,—OC(O)R′, —C(O)R′, —CO₂R′, —CONR′R″, —OC(O)NR′R″, —NR″C(O)R′,—NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″, —NR—C(NR′R″)═NR′″,—S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —NR′NR″R′″, —ONR′R″,—NR′C(O)NR″NR′″R″″, —CN, —NO₂, —R′, —N₃, —CH(Ph)₂, fluoro(C₁-C₄)alkoxy,and fluoro(C₁-C₄)alkyl, —NR′SO₂R″, —NR′C(O)R″, —NR′C(O)—OR″, —NR′OR″, ina number ranging from zero to the total number of open valences on thearomatic ring system; and where R′, R″, R′″, and R″″ are preferablyindependently selected from hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, and substituted or unsubstitutedheteroaryl. When a compound described herein includes more than one Rgroup, for example, each of the R groups is independently selected asare each R′, R″, R′″, and R″″ groups when more than one of these groupsis present.

Substituents for rings (e.g., cycloalkyl, heterocycloalkyl, aryl,heteroaryl, cycloalkylene, heterocycloalkylene, arylene, orheteroarylene) may be depicted as substituents on the ring rather thanon a specific atom of a ring (commonly referred to as a floatingsubstituent). In such a case, the substituent may be attached to any ofthe ring atoms (obeying the rules of chemical valency) and in the caseof fused rings or spirocyclic rings, a substituent depicted asassociated with one member of the fused rings or spirocyclic rings (afloating substituent on a single ring), may be a substituent on any ofthe fused rings or spirocyclic rings (a floating substituent on multiplerings). When a substituent is attached to a ring, but not a specificatom (a floating substituent), and a subscript for the substituent is aninteger greater than one, the multiple substituents may be on the sameatom, same ring, different atoms, different fused rings, differentspirocyclic rings, and each substituent may optionally be different.Where a point of attachment of a ring to the remainder of a molecule isnot limited to a single atom (a floating substituent), the attachmentpoint may be any atom of the ring and in the case of a fused ring orspirocyclic ring, any atom of any of the fused rings or spirocyclicrings while obeying the rules of chemical valency. Where a ring, fusedrings, or spirocyclic rings contain one or more ring heteroatoms and thering, fused rings, or spirocyclic rings are shown with one more floatingsubstituents (including, but not limited to, points of attachment to theremainder of the molecule), the floating substituents may be bonded tothe heteroatoms. Where the ring heteroatoms are shown bound to one ormore hydrogens (e.g., a ring nitrogen with two bonds to ring atoms and athird bond to a hydrogen) in the structure or formula with the floatingsubstituent, when the heteroatom is bonded to the floating substituent,the substituent will be understood to replace the hydrogen, whileobeying the rules of chemical valency.

Two or more substituents may optionally be joined to form aryl,heteroaryl, cycloalkyl, or heterocycloalkyl groups. Such so-calledring-forming substituents are typically, though not necessarily, foundattached to a cyclic base structure. In one embodiment, the ring-formingsubstituents are attached to adjacent members of the base structure. Forexample, two ring-forming substituents attached to adjacent members of acyclic base structure create a fused ring structure. In anotherembodiment, the ring-forming substituents are attached to a singlemember of the base structure. For example, two ring-forming substituentsattached to a single member of a cyclic base structure create aspirocyclic structure. In yet another embodiment, the ring-formingsubstituents are attached to non-adjacent members of the base structure.

Two of the substituents on adjacent atoms of the aryl or heteroaryl ringmay optionally form a ring of the formula -T-C(O)—(CRR′)_(q)—U—, whereinT and U are independently —NR—, —O—, —CRR′—, or a single bond, and q isan integer of from 0 to 3. Alternatively, two of the substituents onadjacent atoms of the aryl or heteroaryl ring may optionally be replacedwith a substituent of the formula -A-(CH₂)_(r)—B—, wherein A and B areindependently —CRR′—, —O—, —NR—, —S—, —S(O)—, —S(O)₂—, —S(O)₂NR′—, or asingle bond, and r is an integer of from 1 to 4. One of the single bondsof the new ring so formed may optionally be replaced with a double bond.Alternatively, two of the substituents on adjacent atoms of the aryl orheteroaryl ring may optionally be replaced with a substituent of theformula —(CRR′)_(s)—X′— (C″R″R′″)_(d)—, where s and d are independentlyintegers of from 0 to 3, and X is —O—, —NR′—, —S—, —S(O)—, —S(O)₂—, or—S(O)₂NR′—. The substituents R, R′, R″, and R′″ are preferablyindependently selected from hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, and substituted or unsubstitutedheteroaryl.

As used herein, the terms “heteroatom” or “ring heteroatom” are meant toinclude oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), andsilicon (Si).

A “substituent group,” as used herein, means a group selected from thefollowing moieties:

-   -   (A) oxo, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CHCl₂, —CHBr₂,        —CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I, —CN, —OH, —NH₂,        —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,        —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH,        —OCCl₃, —OCF₃, —OCBr₃, —OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂,        —OCH₂Cl, —OCH₂Br, —OCH₂I, —OCH₂F, —N₃, unsubstituted alkyl        (e.g., C₁-C₈ alkyl, C₁-C₆ alkyl, or C₁-C₄ alkyl), unsubstituted        heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered        heteroalkyl, or 2 to 4 membered heteroalkyl), unsubstituted        cycloalkyl (e.g., C₃-C₈ cycloalkyl, C₃-C₆ cycloalkyl, or C₅-C₆        cycloalkyl), unsubstituted heterocycloalkyl (e.g., 3 to 8        membered heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or        5 to 6 membered heterocycloalkyl), unsubstituted aryl (e.g.,        C₆-C₁₀ aryl, C₁₀ aryl, or phenyl), or unsubstituted heteroaryl        (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl,        or 5 to 6 membered heteroaryl), and    -   (B) alkyl (e.g., C₁-C₈ alkyl, C₁-C₆ alkyl, or C₁-C₄ alkyl),        heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered        heteroalkyl, or 2 to 4 membered heteroalkyl), cycloalkyl (e.g.,        C₃-C₈ cycloalkyl, C₃-C₆ cycloalkyl, or C₅-C₆ cycloalkyl),        heterocycloalkyl (e.g., 3 to 8 membered heterocycloalkyl, 3 to 6        membered heterocycloalkyl, or 5 to 6 membered heterocycloalkyl),        aryl (e.g., C₆-C₁₀ aryl, C₁₀ aryl, or phenyl), heteroaryl (e.g.,        5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to        6 membered heteroaryl), substituted with at least one        substituent selected from:        -   (i) oxo, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CHCl₂, —CHBr₂,            —CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I, —CN, —OH, —NH₂,            —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂,            —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H,            —NHC(O)OH, —NHOH, —OCCl₃, —OCF₃, —OCBr₃, —OCI₃, —OCHCl₂,            —OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl, —OCH₂Br, —OCH₂I, —OCH₂F,            —N₃, unsubstituted alkyl (e.g., C₁-C₈ alkyl, C₁-C₆ alkyl, or            C₁-C₄ alkyl), unsubstituted heteroalkyl (e.g., 2 to 8            membered heteroalkyl, 2 to 6 membered heteroalkyl, or 2 to 4            membered heteroalkyl), unsubstituted cycloalkyl (e.g., C₃-C₈            cycloalkyl, C₃-C₆ cycloalkyl, or C₅-C₆ cycloalkyl),            unsubstituted heterocycloalkyl (e.g., 3 to 8 membered            heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to            6 membered heterocycloalkyl), unsubstituted aryl (e.g.,            C₆-C₁₀ aryl, C₁₀ aryl, or phenyl), or unsubstituted            heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9            membered heteroaryl, or 5 to 6 membered heteroaryl), and        -   (ii) alkyl (e.g., C₁-C₈ alkyl, C₁-C₆ alkyl, or C₁-C₄ alkyl),            heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6            membered heteroalkyl, or 2 to 4 membered heteroalkyl),            cycloalkyl (e.g., C₃-C₈ cycloalkyl, C₃-C₆ cycloalkyl, or            C₅-C₆ cycloalkyl), heterocycloalkyl (e.g., 3 to 8 membered            heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5 to            6 membered heterocycloalkyl), aryl (e.g., C₆-C₁₀ aryl, C₁₀            aryl, or phenyl), heteroaryl (e.g., 5 to 10 membered            heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered            heteroaryl), substituted with at least one substituent            selected from:            -   (a) oxo, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CHCl₂,                —CHBr₂, —CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I, —CN,                —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H,                —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂,                —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃, —OCF₃,                —OCBr₃, —OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂,                —OCH₂Cl, —OCH₂Br, —OCH₂I, —OCH₂F, —N₃, unsubstituted                alkyl (e.g., C₁-C₈ alkyl, C₁-C₆ alkyl, or C₁-C₄ alkyl),                unsubstituted heteroalkyl (e.g., 2 to 8 membered                heteroalkyl, 2 to 6 membered heteroalkyl, or 2 to 4                membered heteroalkyl), unsubstituted cycloalkyl (e.g.,                C₃-C₈ cycloalkyl, C₃-C₆ cycloalkyl, or C₅-C₆                cycloalkyl), unsubstituted heterocycloalkyl (e.g., 3 to                8 membered heterocycloalkyl, 3 to 6 membered                heterocycloalkyl, or 5 to 6 membered heterocycloalkyl),                unsubstituted aryl (e.g., C₆-C₁₀ aryl, C₁₀ aryl, or                phenyl), or unsubstituted heteroaryl (e.g., 5 to 10                membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to                6 membered heteroaryl), and            -   (b) alkyl (e.g., C₁-C₈ alkyl, C₁-C₆ alkyl, or C₁-C₄                alkyl), heteroalkyl (e.g., 2 to 8 membered heteroalkyl,                2 to 6 membered heteroalkyl, or 2 to 4 membered                heteroalkyl), cycloalkyl (e.g., C₃-C₈ cycloalkyl, C₃-C₆                cycloalkyl, or C₅-C₆ cycloalkyl), heterocycloalkyl                (e.g., 3 to 8 membered heterocycloalkyl, 3 to 6 membered                heterocycloalkyl, or 5 to 6 membered heterocycloalkyl),                aryl (e.g., C₆-C₁₀ aryl, C₁₀ aryl, or phenyl),                heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9                membered heteroaryl, or 5 to 6 membered heteroaryl),                substituted with at least one substituent selected from:                oxo, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CHCl₂, —CHBr₂,                —CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I, —CN, —OH,                —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂,                —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H,                —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃, —OCF₃, —OCBr₃,                —OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl,                —OCH₂Br, —OCH₂I, —OCH₂F, —N₃, unsubstituted alkyl (e.g.,                C₁-C₈ alkyl, C₁-C₆ alkyl, or C₁-C₄ alkyl), unsubstituted                heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6                membered heteroalkyl, or 2 to 4 membered heteroalkyl),                unsubstituted cycloalkyl (e.g., C₃-C₈ cycloalkyl, C₃-C₆                cycloalkyl, or C₅-C₆ cycloalkyl), unsubstituted                heterocycloalkyl (e.g., 3 to 8 membered                heterocycloalkyl, 3 to 6 membered heterocycloalkyl, or 5                to 6 membered heterocycloalkyl), unsubstituted aryl                (e.g., C₆-C₁₀ aryl, C₁₀ aryl, or phenyl), or                unsubstituted heteroaryl (e.g., 5 to 10 membered                heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6                membered heteroaryl).

A “size-limited substituent” or “size-limited substituent group,” asused herein, means a group selected from all of the substituentsdescribed above for a “substituent group,” wherein each substituted orunsubstituted alkyl is a substituted or unsubstituted C₁-C₂₀ alkyl, eachsubstituted or unsubstituted heteroalkyl is a substituted orunsubstituted 2 to 20 membered heteroalkyl, each substituted orunsubstituted cycloalkyl is a substituted or unsubstituted C₃-C₈cycloalkyl, each substituted or unsubstituted heterocycloalkyl is asubstituted or unsubstituted 3 to 8 membered heterocycloalkyl, eachsubstituted or unsubstituted aryl is a substituted or unsubstitutedC₆-C₁₀ aryl, and each substituted or unsubstituted heteroaryl is asubstituted or unsubstituted 5 to 10 membered heteroaryl.

A “lower substituent” or “lower substituent group,” as used herein,means a group selected from all of the substituents described above fora “substituent group,” wherein each substituted or unsubstituted alkylis a substituted or unsubstituted C₁-C₈ alkyl, each substituted orunsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8membered heteroalkyl, each substituted or unsubstituted cycloalkyl is asubstituted or unsubstituted C₃-C₇ cycloalkyl, each substituted orunsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 7membered heterocycloalkyl, each substituted or unsubstituted aryl is asubstituted or unsubstituted phenyl, and each substituted orunsubstituted heteroaryl is a substituted or unsubstituted 5 to 6membered heteroaryl.

In some embodiments, each substituted group described in the compoundsherein is substituted with at least one substituent group. Morespecifically, in some embodiments, each substituted alkyl, substitutedheteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl,substituted aryl, substituted heteroaryl, substituted alkylene,substituted heteroalkylene, substituted cycloalkylene, substitutedheterocycloalkylene, substituted arylene, and/or substitutedheteroarylene described in the compounds herein are substituted with atleast one substituent group. In other embodiments, at least one or allof these groups are substituted with at least one size-limitedsubstituent group. In other embodiments, at least one or all of thesegroups are substituted with at least one lower substituent group.

In other embodiments of the compounds herein, each substituted orunsubstituted alkyl may be a substituted or unsubstituted C₁-C₂₀ alkyl,each substituted or unsubstituted heteroalkyl is a substituted orunsubstituted 2 to 20 membered heteroalkyl, each substituted orunsubstituted cycloalkyl is a substituted or unsubstituted C₃-C₈cycloalkyl, each substituted or unsubstituted heterocycloalkyl is asubstituted or unsubstituted 3 to 8 membered heterocycloalkyl, eachsubstituted or unsubstituted aryl is a substituted or unsubstitutedC₆-C₁₀ aryl, and/or each substituted or unsubstituted heteroaryl is asubstituted or unsubstituted 5 to 10 membered heteroaryl. In someembodiments of the compounds herein, each substituted or unsubstitutedalkylene is a substituted or unsubstituted C₁-C₂₀ alkylene, eachsubstituted or unsubstituted heteroalkylene is a substituted orunsubstituted 2 to 20 membered heteroalkylene, each substituted orunsubstituted cycloalkylene is a substituted or unsubstituted C₃-C₈cycloalkylene, each substituted or unsubstituted heterocycloalkylene isa substituted or unsubstituted 3 to 8 membered heterocycloalkylene, eachsubstituted or unsubstituted arylene is a substituted or unsubstitutedC₆-C₁₀ arylene, and/or each substituted or unsubstituted heteroaryleneis a substituted or unsubstituted 5 to 10 membered heteroarylene.

In some embodiments, each substituted or unsubstituted alkyl is asubstituted or unsubstituted C₁-C₈ alkyl, each substituted orunsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8membered heteroalkyl, each substituted or unsubstituted cycloalkyl is asubstituted or unsubstituted C₃-C₇ cycloalkyl, each substituted orunsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 7membered heterocycloalkyl, each substituted or unsubstituted aryl is asubstituted or unsubstituted phenyl, and/or each substituted orunsubstituted heteroaryl is a substituted or unsubstituted 5 to 6membered heteroaryl. In some embodiments, each substituted orunsubstituted alkylene is a substituted or unsubstituted C₁-C₈ alkylene,each substituted or unsubstituted heteroalkylene is a substituted orunsubstituted 2 to 8 membered heteroalkylene, each substituted orunsubstituted cycloalkylene is a substituted or unsubstituted C₃-C₇cycloalkylene, each substituted or unsubstituted heterocycloalkylene isa substituted or unsubstituted 3 to 7 membered heterocycloalkylene, eachsubstituted or unsubstituted arylene is a substituted or unsubstitutedphenylene, and/or each substituted or unsubstituted heteroarylene is asubstituted or unsubstituted 5 to 6 membered heteroarylene. In someembodiments, the compound (e.g., nucleotide analogue) is a chemicalspecies set forth in the Examples section, claims, embodiments, figures,or tables below.

In embodiments, a substituted or unsubstituted moiety (e.g., substitutedor unsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, substituted orunsubstituted heteroaryl, substituted or unsubstituted alkylene,substituted or unsubstituted heteroalkylene, substituted orunsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, and/orsubstituted or unsubstituted heteroarylene) is unsubstituted (e.g., isan unsubstituted alkyl, unsubstituted heteroalkyl, unsubstitutedcycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl,unsubstituted heteroaryl, unsubstituted alkylene, unsubstitutedheteroalkylene, unsubstituted cycloalkylene, unsubstitutedheterocycloalkylene, unsubstituted arylene, and/or unsubstitutedheteroarylene, respectively). In embodiments, a substituted orunsubstituted moiety (e.g., substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, substituted or unsubstituted heteroaryl,substituted or unsubstituted alkylene, substituted or unsubstitutedheteroalkylene, substituted or unsubstituted cycloalkylene, substitutedor unsubstituted heterocycloalkylene, substituted or unsubstitutedarylene, and/or substituted or unsubstituted heteroarylene) issubstituted (e.g., is a substituted alkyl, substituted heteroalkyl,substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl,substituted heteroaryl, substituted alkylene, substitutedheteroalkylene, substituted cycloalkylene, substitutedheterocycloalkylene, substituted arylene, and/or substitutedheteroarylene, respectively).

In embodiments, a substituted moiety (e.g., substituted alkyl,substituted heteroalkyl, substituted cycloalkyl, substitutedheterocycloalkyl, substituted aryl, substituted heteroaryl, substitutedalkylene, substituted heteroalkylene, substituted cycloalkylene,substituted heterocycloalkylene, substituted arylene, and/or substitutedheteroarylene) is substituted with at least one substituent group,wherein if the substituted moiety is substituted with a plurality ofsubstituent groups, each substituent group may optionally be different.In embodiments, if the substituted moiety is substituted with aplurality of substituent groups, each substituent group is different.

In embodiments, a substituted moiety (e.g., substituted alkyl,substituted heteroalkyl, substituted cycloalkyl, substitutedheterocycloalkyl, substituted aryl, substituted heteroaryl, substitutedalkylene, substituted heteroalkylene, substituted cycloalkylene,substituted heterocycloalkylene, substituted arylene, and/or substitutedheteroarylene) is substituted with at least one size-limited substituentgroup, wherein if the substituted moiety is substituted with a pluralityof size-limited substituent groups, each size-limited substituent groupmay optionally be different. In embodiments, if the substituted moietyis substituted with a plurality of size-limited substituent groups, eachsize-limited substituent group is different.

In embodiments, a substituted moiety (e.g., substituted alkyl,substituted heteroalkyl, substituted cycloalkyl, substitutedheterocycloalkyl, substituted aryl, substituted heteroaryl, substitutedalkylene, substituted heteroalkylene, substituted cycloalkylene,substituted heterocycloalkylene, substituted arylene, and/or substitutedheteroarylene) is substituted with at least one lower substituent group,wherein if the substituted moiety is substituted with a plurality oflower substituent groups, each lower substituent group may optionally bedifferent. In embodiments, if the substituted moiety is substituted witha plurality of lower substituent groups, each lower substituent group isdifferent.

In embodiments, a substituted moiety (e.g., substituted alkyl,substituted heteroalkyl, substituted cycloalkyl, substitutedheterocycloalkyl, substituted aryl, substituted heteroaryl, substitutedalkylene, substituted heteroalkylene, substituted cycloalkylene,substituted heterocycloalkylene, substituted arylene, and/or substitutedheteroarylene) is substituted with at least one substituent group,size-limited substituent group, or lower substituent group; wherein ifthe substituted moiety is substituted with a plurality of groupsselected from substituent groups, size-limited substituent groups, andlower substituent groups; each substituent group, size-limitedsubstituent group, and/or lower substituent group may optionally bedifferent. In embodiments, if the substituted moiety is substituted witha plurality of groups selected from substituent groups, size-limitedsubstituent groups, and lower substituent groups; each substituentgroup, size-limited substituent group, and/or lower substituent group isdifferent.

In a recited claim or chemical formula description herein, each Rsubstituent or L linker that is described as being “substituted” withoutreference as to the identity of any chemical moiety that composes the“substituted” group (also referred to herein as an “open substitution”on an R substituent or L linker or an “openly substituted” R substituentor L linker), the recited R substituent or L linker may, in embodiments,be substituted with one or more first substituent groups as definedbelow.

The first substituent group is denoted with a corresponding firstdecimal point numbering system such that, for example, R¹ may besubstituted with one or more first substituent groups denoted byR^(1.1), R² may be substituted with one or more first substituent groupsdenoted by R^(2.1), R³ may be substituted with one or more firstsubstituent groups denoted by R^(3.1), R⁴ may be substituted with one ormore first substituent groups denoted by R^(4.1), R⁵ may be substitutedwith one or more first substituent groups denoted by R^(5.1), and thelike up to or exceeding an R¹⁰⁰ that may be substituted with one or morefirst substituent groups denoted by R^(100.1). As a further example,R^(1A) may be substituted with one or more first substituent groupsdenoted by R^(1A)—, R^(2A) may be substituted with one or more firstsubstituent groups denoted by R^(2A.1), R^(3A) may be substituted withone or more first substituent groups denoted by R^(3A.1), R^(4A) may besubstituted with one or more first substituent groups denoted byR^(4A.1), R^(5A) may be substituted with one or more first substituentgroups denoted by R^(5A.1) and the like up to or exceeding an R^(100A)may be substituted with one or more first substituent groups denoted byR^(100A.1). As a further example, L¹ may be substituted with one or morefirst substituent groups denoted by R^(L1.1), L² may be substituted withone or more first substituent groups denoted by R^(L2.1), L³ may besubstituted with one or more first substituent groups denoted byR^(L3.1), L⁴ may be substituted with one or more first substituentgroups denoted by R^(L4.1), L⁵ may be substituted with one or more firstsubstituent groups denoted by R^(L5.1) and the like up to or exceedingan L¹⁰⁰ which may be substituted with one or more first substituentgroups denoted by R^(L100.1) Thus, each numbered R group or L group(alternatively referred to herein as R^(WW) or L^(WW) wherein “WW”represents the stated superscript number of the subject R group or Lgroup) described herein may be substituted with one or more firstsubstituent groups referred to herein generally as R^(WW.1) or R^(LWW.1)respectively. In turn, each first substituent group (e.g., R^(1.1),R^(2.1), R^(3.1), R^(4.1), R^(5.1) . . . R^(100.1); R^(1A.1), R^(2A.1),R^(3A.1), R^(4A.1), R^(5A.1) . . . R^(100A.1); R^(L1.1), R^(L2.1),R^(L3.1), R^(L4.1), R^(L5.1) . . . R^(L100.1)) may be furthersubstituted with one or more second substituent groups (e.g., R^(1.2),R^(2.2), R^(3.2), R^(4.2), R^(5.2) . . . R^(100.2); R^(1A.2), R^(2A.2),R^(3A.2), R^(4A.2), R^(5A.2) . . . R^(100A.2); R^(L1.2), R^(L2.2),R^(L3.2), R^(L4.2), R^(L5.2) . . . R^(L100.2), respectively). Thus, eachfirst substituent group, which may alternatively be represented hereinas R^(WW.1) as described above, may be further substituted with one ormore second substituent groups, which may alternatively be representedherein as R^(WW.2).

Finally, each second substituent group (e.g., R^(1.2), R^(2.2), R^(3.2),R^(4.2), R^(5.2) . . . R^(100.2), R^(1A.2), R^(2A.2), R^(3A.2),R^(4A.2), R^(5A.2) . . . R^(100A.2); R^(L1.2), R^(L2.2), R^(L3.2),R^(L4.2), R^(L5.2) . . . R^(L100.2)) may be further substituted with oneor more third substituent groups (e.g., R^(1.3), R^(2.3), R^(3.3),R^(4.3), R^(5.3) . . . R^(100.3). R^(1A.3), R^(2A.3), R^(3A.3),R^(4A.3), R^(5A.3) . . . R^(100A.3); R^(L1.3), R^(L2.3), R^(L3.3),R^(L4.3), R^(L5.3), R^(L100.3); respectively). Thus, each secondsubstituent group, which may alternatively be represented herein asR^(WW.2) as described above, may be further substituted with one or morethird substituent groups, which may alternatively be represented hereinas R^(WW.3). Each of the first substituent groups may be optionallydifferent. Each of the second substituent groups may be optionallydifferent. Each of the third substituent groups may be optionallydifferent.

Thus, as used herein, R^(WW) represents a substituent recited in a claimor chemical formula description herein which is openly substituted. “WW”represents the stated superscript number of the subject R group (1, 2,3, 1A, 2A, 3A, 1B, 2B, 3B, etc.). Likewise, L^(WW) is a linker recitedin a claim or chemical formula description herein which is openlysubstituted. Again, “WW” represents the stated superscript number of thesubject L group (1, 2, 3, 1A, 2A, 3A, 1B, 2B, 3B, etc.). As statedabove, in embodiments, each R^(WW) may be unsubstituted or independentlysubstituted with one or more first substituent groups, referred toherein as R^(WW.1); each first substituent group, R^(WW.1), may beunsubstituted or independently substituted with one or more secondsubstituent groups, referred to herein as R^(WW.2); and each secondsubstituent group may be unsubstituted or independently substituted withone or more third substituent groups, referred to herein as R^(WW.3).Similarly, each L^(WW) linker may be unsubstituted or independentlysubstituted with one or more first substituent groups, referred toherein as R^(LWW.1); each first substituent group, R^(LWW.1), may beunsubstituted or independently substituted with one or more secondsubstituent groups, referred to herein as R^(LWW.2); and each secondsubstituent group may be unsubstituted or independently substituted withone or more third substituent groups, referred to herein as R^(LWW.3).Each first substituent group is optionally different. Each secondsubstituent group is optionally different. Each third substituent groupis optionally different. For example, if R^(WW) is phenyl, the saidphenyl group is optionally substituted by one or more R^(WW.1) groups asdefined herein below, e.g., when R^(WW.1) is R^(WW.2)-substituted orunsubstituted alkyl, examples of groups so formed include but are notlimited to itself optionally substituted by 1 or more R^(WW.2), whichR^(WW.2) is optionally substituted by one or more R^(WW.3). By way ofexample when the R^(WW) group is phenyl substituted by R^(WW.1), whichis methyl, the methyl group may be further substituted to form groupsincluding but not limited to:

R^(WW.1) is independently oxo, halogen, —CX^(WW.1) ₃, —CHX^(WW.1) ₂,—CH₂X^(WW.1), —OCX^(WW.1) ₃, —OCH₂X^(WW.1), —OCHX^(WW.1) ₂, —CN, —OH,—NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC═(O)NHNH₂, —NHC═(O)NH₂, —NHSO₂H, —NHC═(O)H, —NHC(O)—OH, —NHOH, —N₃,R¹²-substituted or unsubstituted alkyl (e.g., C₁-C₈, C₁-C₆, C₁-C₄, orC₁-C₂), R^(WW.2)-substituted or unsubstituted heteroalkyl (e.g., 2 to 8membered, 2 to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4 to 5membered), R^(WW.2)-substituted or unsubstituted cycloalkyl (e.g.,C₃-C₈, C₃-C₆, C₄-C₆, or C₅-C₆), R^(WW.2)-substituted or unsubstitutedheterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6membered, 4 to 5 membered, or 5 to 6 membered), R^(WW.2)-substituted orunsubstituted aryl (e.g., C₆-C₁₂, C₆-C₁₀, or phenyl), orR^(WW.2)-substituted or unsubstituted heteroaryl (e.g., 5 to 12membered, 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). Inembodiments, R^(WW.1) is independently oxo, halogen, —CX^(WW.1) ₃,—CHX^(WW.1) ₂, —CH₂X^(WW.1), —OCX^(WW.1) ₃, —OCH₂X^(WW.1), —OCHX^(WW.1)₂, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂,—NHNH₂, —ONH₂, —NHC═(O)NHNH₂, —NHC═(O)NH₂, —NHSO₂H, —NHC═(O)H,—NHC(O)—OH, —NHOH, —N₃, unsubstituted alkyl (e.g., C₁-C₈, C₁-C₆, C₁-C₄,or C₁-C₂), unsubstituted heteroalkyl (e.g., 2 to 8 membered, 2 to 6membered, 4 to 6 membered, 2 to 3 membered, or 4 to 5 membered),unsubstituted cycloalkyl (e.g., C₃-C₈, C₃-C₆, C₄-C₆, or C₅-C₆),unsubstituted heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered,4 to 6 membered, 4 to 5 membered, or 5 to 6 membered), unsubstitutedaryl (e.g., C₆-C₁₂, C₆-C₁₀, or phenyl), or unsubstituted heteroaryl(e.g., 5 to 12 membered, 5 to 10 membered, 5 to 9 membered, or 5 to 6membered). X^(WW.1) is independently —F, —Cl, —Br, or —I.

R^(WW.2) is independently oxo, halogen, —CX^(WW.2) ₃, —CHX^(WW.2) ₂,—CH₂X^(WW.2), —OCX^(WW.2) ₃, —OCH₂X^(WW.2), —OCHX^(WW.2) ₂, —CN, —OH,—NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC═(O)NHNH₂, —NHC═(O)NH₂, —NHSO₂H, —NHC═(O)H, —NHC(O)—OH, —NHOH, —N₃,R^(WW.3)-substituted or unsubstituted alkyl (e.g., C₁-C₈, C₁-C₆, C₁-C₄,or C₁-C₂), R^(WW.3)-substituted or unsubstituted heteroalkyl (e.g., 2 to8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4 to 5membered), R^(WW.3)-substituted or unsubstituted cycloalkyl (e.g.,C₃-C₈, C₃-C₆, C₄-C₆, or C₅-C₆), R^(WW.3)-substituted or unsubstitutedheterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6membered, 4 to 5 membered, or 5 to 6 membered), R^(WW.3)-substituted orunsubstituted aryl (e.g., C₆-C₁₂, C₆-C₁₀, or phenyl), orR^(WW.3)-substituted or unsubstituted heteroaryl (e.g., 5 to 12membered, 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). Inembodiments, R^(WW.2) is independently oxo, halogen, —CX^(WW.2) ₃,—CHX^(WW.2) ₂, —CH₂X^(WW.2), —OCX^(WW.2) ₃, —OCH₂X^(WW.2), —OCHX^(WW.2)₂, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂,—NHNH₂, —ONH₂, —NHC═(O)NHNH₂, —NHC═(O)NH₂, —NHSO₂H, —NHC═(O)H,—NHC(O)—OH, —NHOH, —N₃, unsubstituted alkyl (e.g., C₁-C₈, C₁-C₆, C₁-C₄,or C₁-C₂), unsubstituted heteroalkyl (e.g., 2 to 8 membered, 2 to 6membered, 4 to 6 membered, 2 to 3 membered, or 4 to 5 membered),unsubstituted cycloalkyl (e.g., C₃-C₈, C₃-C₆, C₄-C₆, or C₅-C₆),unsubstituted heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered,4 to 6 membered, 4 to 5 membered, or 5 to 6 membered), unsubstitutedaryl (e.g., C₆-C₁₂, C₆-C₁₀, or phenyl), or unsubstituted heteroaryl(e.g., 5 to 12 membered, 5 to 10 membered, 5 to 9 membered, or 5 to 6membered). X^(WW.2) is independently —F, —Cl, —Br, or —I.

R^(WW.3) is independently oxo, halogen, —CX^(WW.3) ₃, —CHX^(WW.3) ₂,—CH₂X^(WW.3), —OCX^(WW.3) ₃, —OCH₂X^(WW.3), —OCHX^(WW.3) ₂, —CN, —OH,—NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC═(O)NHNH₂, —NHC═(O)NH₂, —NHSO₂H, —NHC═(O)H, —NHC(O)—OH, —NHOH, —N₃,unsubstituted alkyl (e.g., C₁-C₈, C₁-C₆, C₁-C₄, or C₁-C₂), unsubstitutedheteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2to 3 membered, or 4 to 5 membered), unsubstituted cycloalkyl (e.g.,C₃-C₈, C₃-C₆, C₄-C₆, or C₅-C₆), unsubstituted heterocycloalkyl (e.g., 3to 8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5to 6 membered), unsubstituted aryl (e.g., C₆-C₁₂, C₆—C₁₀, or phenyl), orunsubstituted heteroaryl (e.g., 5 to 12 membered, 5 to 10 membered, 5 to9 membered, or 5 to 6 membered). X^(WW.3) is independently —F, —Cl, —Br,or —I.

Where two different R^(WW) substituents are joined together to form anopenly substituted ring (e.g., substituted cycloalkyl, substitutedheterocycloalkyl, substituted aryl or substituted heteroaryl), inembodiments the openly substituted ring may be independently substitutedwith one or more first substituent groups, referred to herein asR^(WW.1); each first substituent group, R^(WW.1), may be unsubstitutedor independently substituted with one or more second substituent groups,referred to herein as R^(WW.2); and each second substituent group,R^(WW.2), may be unsubstituted or independently substituted with one ormore third substituent groups, referred to herein as R^(WW.3); and eachthird substituent group, R^(WW.3), is unsubstituted. Each firstsubstituent group is optionally different. Each second substituent groupis optionally different. Each third substituent group is optionallydifferent. In the context of two different R^(WW) substituents joinedtogether to form an openly substituted ring, the “WW” symbol in theR^(WW.1), R^(WW.2) and R^(WW.3) refers to the designated number of oneof the two different R^(WW) substituents. For example, in embodimentswhere R^(100A) and R^(100B) are optionally joined together to form anopenly substituted ring, R^(WW.1) is R^(100B.1), R^(WW.2) is R^(100A.2),and R^(WW.3) is R^(100A.3). Alternatively, in embodiments where R^(100A)and R^(100B) are optionally joined together to form an openlysubstituted ring, R^(WW.1) is R^(100B.1), R^(WW.2) is R^(100B.2), andR^(WW.3) is R^(100B.3). R^(WW.1), R^(WW.2) and R^(WW.3) in thisparagraph are as defined in the preceding paragraphs.

R^(LWW.1) is independently oxo, halogen, —CX^(LWW.1) ₃, —CHX^(LWW.1) ₂,—CH₂X^(LWW.1), —OCX^(LWW.1) ₃, —OCH₂X^(LWW.1), —OCHX^(LWW.1) ₂, —CN,—OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂,—ONH₂, —NHC═(O)NHNH₂, —NHC═(O)NH₂, —NHSO₂H, —NHC═(O)H, —NHC(O)—OH,—NHOH, —N₃, R^(LWW.2)_substituted or unsubstituted alkyl (e.g., C₁-C₈,C₁-C₆, C₁-C₄, or C₁-C₂), R^(LWW.2)-substituted or unsubstitutedheteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2to 3 membered, or 4 to 5 membered), R^(LWW.2)-substituted orunsubstituted cycloalkyl (e.g., C₃-C₈, C₃-C₆, C₄-C₆, or C₅-C₆),R^(LWW.2)-substituted or unsubstituted heterocycloalkyl (e.g., 3 to 8membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6membered), R^(LWW.2)_substituted or unsubstituted aryl (e.g., C₆-C₁₂,C₆-C₁₀, or phenyl), or R^(LWW.2)_substituted or unsubstituted heteroaryl(e.g., 5 to 12 membered, 5 to 10 membered, 5 to 9 membered, or 5 to 6membered). In embodiments, R^(LWW.1) is independently oxo, halogen,—CX^(LWW.1) ₃, —CHX^(LWW.1) ₂, —CH₂X^(LWW.1), —OCX^(LWW.1) ₃,—OCH₂X^(LWW.1), —OCHX^(LWW.1) ₂, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂,—SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂, —NHC═(O) NH₂,—NHSO₂H, —NHC═(O)H, —NHC(O)—OH, —NHOH, —N₃, unsubstituted alkyl (e.g.,C₁-C₈, C₁-C₆, C₁-C₄, or C₁-C₂), unsubstituted heteroalkyl (e.g., 2 to 8membered, 2 to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4 to 5membered), unsubstituted cycloalkyl (e.g., C₃-C₈, C₃-C₆, C₄-C₆, orC₅-C₆), unsubstituted heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered),unsubstituted aryl (e.g., C₆-C₁₂, C₆-C₁₀, or phenyl), or unsubstitutedheteroaryl (e.g., 5 to 12 membered, 5 to 10 membered, 5 to 9 membered,or 5 to 6 membered). X^(LWW.1) is independently —F, —Cl, —Br, or —I.

R^(LWW.2) is independently oxo, halogen, —CX^(LWW.2) ₃, —CHX^(LWW.2) ₂,—CH₂X^(LWW.2), —OCX^(LWW.2) ₃, —OCH₂X^(LWW.2), —OCHX^(LWW.2) ₂, —CN,—OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂,—ONH₂, —NHC═(O)NHNH₂, —NHC═(O)NH₂, —NHSO₂H, —NHC═(O)H, —NHC(O)—OH,—NHOH, —N₃, R^(LWW.3)-substituted or unsubstituted alkyl (e.g., C₁-C₈,C₁-C₆, C₁-C₄, or C₁-C₂), R^(LWW.3)-substituted or unsubstitutedheteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2to 3 membered, or 4 to 5 membered), R^(WW.3)-substituted orunsubstituted cycloalkyl (e.g., C₃-C₈, C₃-C₆, C₄-C₆, or C₅-C₆),R^(LWW.3)-substituted or unsubstituted heterocycloalkyl (e.g., 3 to 8membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6membered), R^(LWW.3)-substituted or unsubstituted aryl (e.g., C₆-C₁₂,C₆-C₁₀, or phenyl), or R^(LWW.3)-substituted or unsubstituted heteroaryl(e.g., 5 to 12 membered, 5 to 10 membered, 5 to 9 membered, or 5 to 6membered). In embodiments, R^(LWW.2) is independently oxo, halogen,—CX^(LWW.2) ₃, —CHX^(LWW.2) ₂, —CH₂X^(LWW.2), —OCX^(LWW.2) ₃,—OCH₂X^(LWW.2), —OCHX^(LWW.2) ₂, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂,—SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC═(O)NHNH₂, —NHC═(O)NH₂,—NHSO₂H, —NHC═(O)H, —NHC(O)—OH, —NHOH, —N₃, unsubstituted alkyl (e.g.,C₁-C₈, C₁-C₆, C₁-C₄, or C₁-C₂), unsubstituted heteroalkyl (e.g., 2 to 8membered, 2 to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4 to 5membered), unsubstituted cycloalkyl (e.g., C₃-C₈, C₃-C₆, C₄-C₆, orC₅-C₆), unsubstituted heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered),unsubstituted aryl (e.g., C₆-C₁₂, C₆-C₁₀, or phenyl), or unsubstitutedheteroaryl (e.g., 5 to 12 membered, 5 to 10 membered, 5 to 9 membered,or 5 to 6 membered). X^(LWW.2) is independently —F, —Cl, —Br, or —I.

R^(LWW.3) is independently oxo, halogen, —CX^(LWW.3) ₃, —CHX^(LWW.3) ₂,—CH₂X^(LWW.3), —OCX^(LWW.3) ₃, —OCH₂X^(LWW.3), —OCHX^(LWW.3) ₂, —CN,—OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂,—ONH₂, —NHC═(O)NHNH₂, —NHC═(O)NH₂, —NHSO₂H, —NHC═(O)H, —NHC(O)—OH,—NHOH, —N₃, unsubstituted alkyl (e.g., C₁-C₈, C₁-C₆, C₁-C₄, or C₁-C₂),unsubstituted heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered, 4 to6 membered, 2 to 3 membered, or 4 to 5 membered), unsubstitutedcycloalkyl (e.g., C₃-C₈, C₃-C₆, C₄-C₆, or C₅-C₆), unsubstitutedheterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6membered, 4 to 5 membered, or 5 to 6 membered), unsubstituted aryl(e.g., C₆-C₁₂, C₆-C₁₀, or phenyl), or unsubstituted heteroaryl (e.g., 5to 12 membered, 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered).X^(LWW.3) is independently —F, —Cl, —Br, or —I.

In the event that any R group recited in a claim or chemical formuladescription set forth herein (R″ substituent) is not specificallydefined in this disclosure, then that R group (R″ group) is herebydefined as independently oxo, halogen, —CX^(WW) ₃, —CHX^(WW) ₂,—CH₂X^(WW), —OCX^(WW) ₃, —OCH₂X^(WW), —OCHX^(WW) ₂, —CN, —OH, —NH₂,—COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC═(O)NHNH₂, —NHC═(O)NH₂, —NHSO₂H, —NHC═(O)H, —NHC(O)—OH, —NHOH, —N₃,R^(WW.1)-substituted or unsubstituted alkyl (e.g., C₁-C₈, C₁-C₆, C₁-C₄,or C₁-C₂), R^(WW.1)-substituted or unsubstituted heteroalkyl (e.g., 2 to8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4 to 5membered), R^(WW.1)-substituted or unsubstituted cycloalkyl (e.g.,C₃-C₈, C₃-C₆, C₄-C₆, or C₅-C₆), R^(WW.1)-substituted or unsubstitutedheterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6membered, 4 to 5 membered, or 5 to 6 membered), R^(WW.1)-substituted orunsubstituted aryl (e.g., C₆-C₁₂, C₆-C₁₀, or phenyl), orR^(WW.1)-substituted or unsubstituted heteroaryl (e.g., 5 to 12membered, 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). X^(WW)is independently —F, —Cl, —Br, or —I. Again, “WW” represents the statedsuperscript number of the subject R group (e.g., 1, 2, 3, 1A, 2A, 3A,1B, 2B, 3B, etc.). R^(WW.1), R^(WW.2), and R^(WW.3) are as definedabove.

In the event that any L linker group recited in a claim or chemicalformula description set forth herein (i.e., an L^(WW) substituent) isnot explicitly defined, then that L group (L^(WW) group) is hereindefined as independently a bond, —O—, —NH—, —C(O)—, —C(O)NH—, —NHC(O)—,—NHC(O)NH—, —C(O)O—, —OC(O)—, —S—, —SO₂NH—, R^(LWW.1)-substituted orunsubstituted alkylene (e.g., C₁-C₈, C₁-C₆, C₁-C₄, or C₁-C₂),R^(LWW.1)-substituted or unsubstituted heteroalkylene (e.g., 2 to 8membered, 2 to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4 to 5membered), R^(LWW.1)-substituted or unsubstituted cycloalkylene (e.g.,C₃-C₈, C₃-C₆, C₄-C₆, or C₅-C₆), R^(LWW.1)-substituted or unsubstitutedheterocycloalkylene (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6membered, 4 to 5 membered, or 5 to 6 membered), R^(LWW.1)-substituted orunsubstituted arylene (e.g., C₆-C₁₂, C₆-C₁₀, or phenyl), orR^(LWW.1)-substituted or unsubstituted heteroarylene (e.g., 5 to 12membered, 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered). Again,“WW” represents the stated superscript number of the subject L group (1,2, 3, 1A, 2A, 3A, 1B, 2B, 3B, etc.). R^(LWW.1), as well as R^(LWW.2) andR^(WW.3) are as defined above.

Certain compounds of the present disclosure possess asymmetric carbonatoms (optical or chiral centers) or double bonds; the enantiomers,racemates, diastereomers, tautomers, geometric isomers, stereoisomericforms that may be defined, in terms of absolute stereochemistry, as (R)-or (S)- or, as (D)- or (L)- for amino acids, and individual isomers areencompassed within the scope of the present disclosure. The compounds ofthe present disclosure do not include those that are known in art to betoo unstable to synthesize and/or isolate. The present disclosure ismeant to include compounds in racemic and optically pure forms.Optically active (R)- and (S)-, or (D)- and (L)-isomers may be preparedusing chiral synthons or chiral reagents, or resolved using conventionaltechniques. When the compounds described herein contain olefinic bondsor other centers of geometric asymmetry, and unless specified otherwise,it is intended that the compounds include both E and Z geometricisomers.

As used herein, the term “isomers” refers to compounds having the samenumber and kind of atoms, and hence the same molecular weight, butdiffering in respect to the structural arrangement or configuration ofthe atoms.

The term “tautomer,” as used herein, refers to one of two or morestructural isomers which exist in equilibrium and which are readilyconverted from one isomeric form to another.

It will be apparent to one skilled in the art that certain compounds ofthis disclosure may exist in tautomeric forms, all such tautomeric formsof the compounds being within the scope of the disclosure.

Unless otherwise stated, structures depicted herein are also meant toinclude all stereochemical forms of the structure; i.e., the R and Sconfigurations for each asymmetric center. Therefore, singlestereochemical isomers as well as enantiomeric and diastereomericmixtures of the present compounds are within the scope of thedisclosure.

Unless otherwise stated, structures depicted herein are also meant toinclude compounds which differ only in the presence of one or moreisotopically enriched atoms. For example, compounds having the presentstructures except for the replacement of a hydrogen by a deuterium ortritium, or the replacement of a carbon by ¹³C- or ¹⁴C-enriched carbonare within the scope of this disclosure.

The compounds of the present disclosure may also contain unnaturalproportions of atomic isotopes at one or more of the atoms thatconstitute such compounds. For example, the compounds may beradiolabeled with radioactive isotopes, such as for example tritium(³H), iodine-125 (¹²⁵I), or carbon-14 (¹⁴C). All isotopic variations ofthe compounds of the present disclosure, whether radioactive or not, areencompassed within the scope of the present disclosure.

It should be noted that throughout the application that alternatives arewritten in Markush groups, for example, each amino acid position thatcontains more than one possible amino acid. It is specificallycontemplated that each member of the Markush group should be consideredseparately, thereby comprising another embodiment, and the Markush groupis not to be read as a single unit.

“Analog,” “analogue” or “derivative” is used in accordance with itsplain ordinary meaning within Chemistry and Biology and refers to achemical compound that is structurally similar to another compound(i.e., a so-called “reference” compound) but differs in composition,e.g., in the replacement of one atom by an atom of a different element,or in the presence of a particular functional group, or the replacementof one functional group by another functional group, or the absolutestereochemistry of one or more chiral centers of the reference compound.Accordingly, an analog is a compound that is similar or comparable infunction and appearance but not in structure or origin to a referencecompound.

The phrase “substituted with a[n],” as used herein, means the specifiedgroup may be substituted with one or more of any or all of the namedsubstituents. For example, where a group, such as an alkyl or heteroarylgroup, is “substituted with an unsubstituted C₁-C₂₀ alkyl, orunsubstituted 2 to 20 membered heteroalkyl,” the group may contain oneor more unsubstituted C₁-C₂₀ alkyls, and/or one or more unsubstituted 2to 20 membered heteroalkyls.

Moreover, where a moiety is substituted with an R substituent, the groupmay be referred to as “R-substituted.” Where a moiety is R-substituted,the moiety is substituted with at least one R substituent and each Rsubstituent is optionally different. Where a particular R group ispresent in the description of a chemical genus (such as Formula (I)), aRoman alphabetic symbol may be used to distinguish each appearance ofthat particular R group. For example, where multiple R¹³ substituentsare present, each R¹³ substituent may be distinguished as R^(13A),R^(13B), R^(13C), R^(13D), etc., wherein each of R^(13A), R^(13B),R^(13C), R^(13D), etc. is defined within the scope of the definition ofR¹³ and optionally differently.

Descriptions of compounds (e.g., polymers) of the present disclosure arelimited by principles of chemical bonding known to those skilled in theart. Accordingly, where a group may be substituted by one or more of anumber of substituents, such substitutions are selected so as to complywith principles of chemical bonding and to give compounds which are notinherently unstable and/or would be known to one of ordinary skill inthe art as likely to be unstable under ambient conditions, such asaqueous, neutral, and several known physiological conditions. Forexample, a heterocycloalkyl or heteroaryl is attached to the remainderof the molecule via a ring heteroatom in compliance with principles ofchemical bonding known to those skilled in the art thereby avoidinginherently unstable compounds.

Certain compounds of the present invention can exist in unsolvated formsas well as solvated forms, including hydrated forms. In general, thesolvated forms are equivalent to unsolvated forms and are encompassedwithin the scope of the present invention. Certain compounds of thepresent invention may exist in multiple crystalline or amorphous forms.In general, all physical forms are equivalent for the uses contemplatedby the present invention and are intended to be within the scope of thepresent invention.

The terms “bioconjugate group,” “bioconjugate reactive moiety,” and“bioconjugate reactive group” refer to a chemical moiety whichparticipates in a reaction to form a bioconjugate linker (e.g., covalentlinker). Non-limiting examples of bioconjugate groups include —NH₂,—COOH, —COOCH₃, —N-hydroxysuccinimide, -maleimide,

In embodiments, the bioconjugate reactive group may be protected (e.g.,with a protecting group). In embodiments, the bioconjugate reactivemoiety is —N₃, -DBCO, alkynyl,

or —NH₂. Additional examples of bioconjugate reactive groups and theresulting bioconjugate reactive linkers may be found in the BioconjugateTable below:

Bioconjugate reactive group 1 Bioconjugate reactive group 2 (e.g.,electrophilic bioconjugate (e.g., nucleophilic bioconjugate ResultingBioconjugate reactive moiety) reactive moiety) reactive linker activatedesters amines/anilines carboxamides acrylamides thiols thioethers acylazides amines/anilines carboxamides acyl halides amines/anilinescarboxamides acyl halides alcohols/phenols esters acyl nitrilesalcohols/phenols esters acyl nitriles amines/anilines carboxamidesaldehydes amines/anilines imines aldehydes or ketones hydrazineshydrazones aldehydes or ketones hydroxylamines oximes alkyl halidesamines/anilines alkyl amines alkyl halides carboxylic acids esters alkylhalides thiols thioethers alkyl halides alcohols/phenols ethers alkylsulfonates thiols thioethers alkyl sulfonates carboxylic acids estersalkyl sulfonates alcohols/phenols ethers anhydrides alcohols/phenolsesters anhydrides amines/anilines carboxamides aryl halides thiolsthiophenols aryl halides amines aryl amines aziridines thiols thioethersboronates glycols boronate esters carbodiimides carboxylic acidsN-acylureas or anhydrides diazoalkanes carboxylic acids esters epoxidesthiols thioethers haloacetamides thiols thioethers haloplatinate aminoplatinum complex haloplatinate heterocycle platinum complexhaloplatinate thiol platinum complex halotriazines amines/anilinesaminotri azines halotriazines alcohols/phenols triazinyl ethershalotriazines thiols triazinyl thioethers imido esters amines/anilinesamidines isocyanates amines/anilines ureas isocyanates alcohols/phenolsurethanes isothiocyanates amines/anilines thioureas maleimides thiolsthioethers phosphoramidites alcohols phosphite esters silyl halidesalcohols silyl ethers sulfonate esters amines/anilines alkyl aminessulfonate esters thiols thioethers sulfonate esters carboxylic acidsesters sulfonate esters alcohols ethers sulfonyl halides amines/anilinessulfonamides sulfonyl halides phenols/alcohols sulfonate esters

As used herein, the term “bioconjugate reactive moiety” and“bioconjugate reactive group” refers to a moiety or group capable offorming a bioconjugate linker (e.g., covalent linker) as a result of theassociation between atoms or molecules of bioconjugate reactive groups.The association can be direct or indirect. For example, a conjugatebetween a first bioconjugate reactive group (e.g., —NH₂, —COOH,—N-hydroxysuccinimide, or -maleimide) and a second bioconjugate reactivegroup (e.g., sulfhydryl, sulfur-containing amino acid, amine, aminesidechain containing amino acid, or carboxylate) provided herein can bedirect, e.g., by covalent bond or linker (e.g., a first linker of secondlinker), or indirect, e.g., by non-covalent bond (e.g., electrostaticinteractions (e.g., ionic bond, hydrogen bond, halogen bond), van derWaals interactions (e.g., dipole-dipole, dipole-induced dipole, Londondispersion), ring stacking (pi effects), hydrophobic interactions andthe like). In embodiments, bioconjugates or bioconjugate linkers areformed using bioconjugate chemistry (i.e., the association of twobioconjugate reactive groups) including, but are not limited tonucleophilic substitutions (e.g., reactions of amines and alcohols withacyl halides, active esters), electrophilic substitutions (e.g., enaminereactions) and additions to carbon-carbon and carbon-heteroatom multiplebonds (e.g., Michael reaction, Diels-Alder addition). These and otheruseful reactions are discussed in, for example, March, ADVANCED ORGANICCHEMISTRY, 3rd Ed., John Wiley & Sons, New York, 1985; Hermanson,BIOCONJUGATE TECHNIQUES, Academic Press, San Diego, 1996; and Feeney etal., MODIFICATION OF PROTEINS; Advances in Chemistry Series, Vol. 198,American Chemical Society, Washington, D.C., 1982. In embodiments, thefirst bioconjugate reactive group (e.g., maleimide moiety) is covalentlyattached to the second bioconjugate reactive group (e.g., a sulfhydryl).In embodiments, the first bioconjugate reactive group (e.g., haloacetylmoiety) is covalently attached to the second bioconjugate reactive group(e.g., a sulfhydryl). In embodiments, the first bioconjugate reactivegroup (e.g., pyridyl moiety) is covalently attached to the secondbioconjugate reactive group (e.g., a sulfhydryl). In embodiments, thefirst bioconjugate reactive group (e.g., —N-hydroxysuccinimide moiety)is covalently attached to the second bioconjugate reactive group (e.g.,an amine). In embodiments, the first bioconjugate reactive group (e.g.,maleimide moiety) is covalently attached to the second bioconjugatereactive group (e.g., a sulfhydryl). In embodiments, the firstbioconjugate reactive group (e.g., -sulfo-N-hydroxysuccinimide moiety)is covalently attached to the second bioconjugate reactive group (e.g.,an amine). In embodiments, a bioconjugate linker is formed by thereaction between an azide moiety and a dibenzocyclooctyne (DBCO) moiety.

Useful bioconjugate reactive groups used for bioconjugate chemistriesherein include, for example: (a) carboxyl groups and various derivativesthereof including, but not limited to, N-hydroxysuccinimide esters,N-hydroxybenztriazole esters, acid halides, acyl imidazoles, thioesters,p-nitrophenyl esters, alkyl, alkenyl, alkynyl and aromatic esters; (b)hydroxyl groups which can be converted to esters, ethers, aldehydes,etc.; (c) haloalkyl groups wherein the halide can be later displacedwith a nucleophilic group such as, for example, an amine, a carboxylateanion, thiol anion, carbanion, or an alkoxide ion, thereby resulting inthe covalent attachment of a new group at the site of the halogen atom;(d) dienophile groups which are capable of participating in Diels-Alderreactions such as, for example, maleimido or maleimide groups; (e)aldehyde or ketone groups such that subsequent derivatization ispossible via formation of carbonyl derivatives such as, for example,imines, hydrazones, semicarbazones or oximes, or via such mechanisms asGrignard addition or alkyllithium addition; (f) sulfonyl halide groupsfor subsequent reaction with amines, for example, to form sulfonamides;(g) thiol groups, which can be converted to disulfides, reacted withacyl halides, or bonded to metals such as gold, or react withmaleimides; (h) amine or sulfhydryl groups (e.g., present in cysteine),which can be, for example, acylated, alkylated or oxidized; (i) alkenes,which can undergo, for example, cycloadditions, acylation, Michaeladdition, etc.; (j) epoxides, which can react with, for example, aminesand hydroxyl compounds; (k) phosphoramidites and other standardfunctional groups useful in nucleic acid synthesis; (l) metal siliconoxide bonding; (m) metal bonding to reactive phosphorus groups (e.g.,phosphines) to form, for example, phosphate diester bonds; (n) azidescoupled to alkynes using copper catalyzed cycloaddition click chemistry;(o) biotin conjugate can react with avidin or strepavidin to form aavidin-biotin complex or streptavidin-biotin complex.

The term “covalent linker” is used in accordance with its ordinarymeaning and refers to a divalent moiety which connects at least twomoieties to form a molecule.

The term “non-covalent linker” is used in accordance with its ordinarymeaning and refers to a divalent moiety which includes at least twomolecules that are not covalently linked to each other but are capableof interacting with each other via a non-covalent bond (e.g.,electrostatic interactions (e.g., ionic bond, hydrogen bond, halogenbond) or van der Waals interactions (e.g., dipole-dipole, dipole-induceddipole, London dispersion). In embodiments, the non-covalent linker isthe result of two molecules that are not covalently linked to each otherthat interact with each other via a non-covalent bond.

The term “non-reactive moiety” is used in accordance with its plainordinary meaning and refers to a moiety that does not react with anucleophile or an electrophile (e.g., under reaction conditions whereinother moieties in the same molecule may react with a nucleophile orelectrophile, under click chemistry reaction conditions such as thoseconditions wherein an azide may react with dibenzocyclooctyne (DBCO) oran epoxide). In embodiments, the non-reactive moiety is attached to apolymer. In embodiments, the non-reactive moiety is hydrophilic. Inembodiments, the non-reactive moiety increases the water solubility of apolymer that includes the non-reactive moiety. In embodiments, thenon-reactive moiety is not a bioconjugate reactive moiety. Inembodiments, the non-reactive moiety is an unsubstituted alkyl. Inembodiments, the non-reactive moiety is hydrogen.

“Contacting” is used in accordance with its plain ordinary meaning andrefers to the process of allowing at least two distinct species (e.g.,chemical compounds including biomolecules, particles, solid supports, orcells) to become sufficiently proximal to react, interact or physicallytouch. It should be appreciated, however, that the resulting reactionproduct can be produced directly from a reaction between the addedreagents or from an intermediate from one or more of the added reagentswhich can be produced in the reaction mixture. The term “contacting” mayinclude allowing two species to react, interact, or physically touch,wherein the two species may be a compound as described herein and aprotein or enzyme. In some embodiments contacting includes allowing aparticle described herein to interact with an array.

The terms “particle” and “bead” are used interchangeably and mean asmall body made of a rigid or semi-rigid material. The body can have ashape characterized, for example, as a sphere, oval, microsphere, orother recognized particle shape whether having regular or irregulardimensions. A “nanoparticle,” as used herein, is a particle wherein thelongest diameter is less than or equal to 1000 nanometers. Nanoparticlesmay be composed of any appropriate material. For example, nanoparticlecores may include appropriate metals and metal oxides thereof (e.g., ametal nanoparticle core), carbon (e.g., an organic nanoparticle core)silicon and oxides thereof (e.g., a silicon nanoparticle core) or boronand oxides thereof (e.g., a boron nanoparticle core), or mixturesthereof. Nanoparticles may be composed of at least two distinctmaterials, one material (e.g., silica) forms the core and the othermaterial forms the shell (e.g., copolymer) surrounding the core. Inembodiments, the nanoparticle is composed of a copolymer describedherein.

The term “silica nanoparticle” is used according to its plain andordinary meaning and refers to a nanoparticle containing Si atoms (e.g.,in a tetrahedral coordination) with 4 oxygen atoms surrounding a centralSi atom. A person of ordinary skill in the art would recognize that thesilica nanoparticle typically includes terminal oxygen atoms (e.g., theoxygens on the surface of the nanoparticle) that are hydroxyl moieties.A silica nanoparticle is a particle wherein the longest diameter istypically less than or equal to 1000 nanometers comprising a matrix ofsilicon-oxygen bonds. In embodiments, a nanoparticle has a shortestdiameter greater than or equal to 1 nanometer (e.g., diameter from 1 to1000 nanometers). In embodiments, the silica nanoparticle is mesoporous.In embodiments, the silica nanoparticle is nonporous.

A functionalized particle, as used herein, may refer to the post hocconjugation (i.e. conjugation after the formation of the particle) of amoiety to a functional group on the surface of a particle. For example,a silica particle may be further functionalized to include additionalatoms (e.g., nitrogen) or chemical entities (e.g., polymeric moieties orbioconjugate group). For example, when the silica nanoparticle isfurther functionalized with a nitrogen containing compound, one of thesurface oxygen atoms surrounding the Si atom may be replaced with anitrogen containing moiety. For example, a silica particle may befunctionalized by reacting an unmodified silica nanoparticle with APTMS,APTES, or AHAMTES to generate an amine functionalized silica particle.The amine group may serve as a bioconjugate reactive moiety. In contrastto a functionalized particle, an unmodified particle refers to aparticle which has not been further functionalized. Thus, for example,an unmodified particle does not include a nitrogen containing moiety(e.g., terminal amine moieties). For example, an unmodified silicananoparticle refers to a silica nanoparticle as synthesized without posthoc functionalization. As used herein, the terms “bare particle” and“unmodified particle” are synonymous and interchangeable. Inembodiments, an unmodified silica nanoparticle includes terminal oxygenatoms (e.g., the oxygens on the surface of the nanoparticle) that arehydroxyl moieties. In embodiments, the terminal oxygen atoms of theunmodified silica nanoparticle are —OH or salts thereof (e.g. —O—moieties). In contrast to a functionalized nanoparticle, an unmodifiednanoparticle refers to a nanoparticle which has not been furtherfunctionalized. Thus, for example, an unmodified silica nanoparticledoes not include a polymeric moiety.

Lengths and sizes of nanoparticles and functionalized particles asdescribed herein may be measured using Transmission Electron Microscopy.For example, transmission electron microscopy measurements of thevarious particle samples may be drop coated (5 μL) onto 200 mesh copperEM grids, air-dried and imaged using a FEI Tecnai 12 TEM equipped with aGatan Ultrascan 2K CCD camera at an accelerating voltage of 120 kV. Theaverage size distributions of the particles may then be obtained fromthe TEM images using Image J software that were plotted using software(e.g., Origin Pro 8) to obtain the histogram size distributions of theparticles. In embodiment, the length of a nanoparticle refers to thelongest dimension of the particle.

As used herein, a “plurality” refers to two or more.

As used herein, “capable of hybridizing” is used in accordance with itsordinary meaning in the art and refers to two oligonucleotides that,under suitable conditions, can form a duplex (e.g., Watson-Crickpairing) which includes a double-stranded portion of nucleic acid. Suchconditions, known in the art and described herein, depend upon, forexample, the nature of the nucleotide sequence, temperature, and bufferconditions. The stringency of hybridization can be influenced by variousparameters, including degree of identity and/or complementarity betweenthe polynucleotides (or any target sequences within the polynucleotides)to be hybridized; melting point of the polynucleotides and/or targetsequences to be hybridized, referred to as “Tm”; parameters such assalts, buffers, pH, temperature, GC % content of the polynucleotide andprimers, and/or time. Typically, hybridization is favored in lowertemperatures and/or increased salt concentrations, as well as reducedconcentrations of organic solvents. Some exemplary conditions suitablefor hybridization include incubation of the polynucleotides to behybridized in solutions having sodium salts, such as NaCl, sodiumcitrate and/or sodium phosphate. In some embodiments, hybridization orwash solutions can include about 10-75% formamide and/or about 0.01-0.7%sodium dodecyl sulfate (SDS). In some embodiments, a hybridizationsolution can be a stringent hybridization solution which can include anycombination of 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodiumcitrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate,5×Denhardt's solution, 0.1% SDS, and/or 10% dextran sulfate. In someembodiments, the hybridization or washing solution can include BSA(bovine serum albumin). In some embodiments, hybridization or washingcan be conducted at a temperature range of about 20-25° C., or about25-30° C., or about 30-35° C., or about 35-40° C., or about 40-45° C.,or about 45-50° C., or about 50-55° C., or higher. In some embodiments,hybridization or washing can be conducted for a time range of about 1-10minutes, or about 10-20 minutes, or about 20-30 minutes, or about 30-40minutes, or about 40-50 minutes, or about 50-60 minutes, or longer. Insome embodiments, hybridization or wash conditions can be conducted at apH range of about 5-10, or about pH 6-9, or about pH 6.5-8, or about pH6.5-7.

The term “alkoxysilyl” as used herein refers to silicon atom covalentlybound to one or more alkoxy groups. In embodiments, the alkoxysilylmoiety has the formula —(R)_(n)—Si(—O-alkyl)₃ moiety, wherein n is 1, 2,or 3 and R is an unsubstituted C₁-C₆ alkyl. When used in combinationwith a polymerizable monomer (e.g., acrylate, methacrylate, acrylamide),it is understood the polymerizable monomer is covalently linked to thealkoxysilyl moiety. For example, alkoxysilyl methacrylate has theformula

wherein L¹ is substituted or unsubstituted alkylene or substituted orunsubstituted heteroalkylene as described herein. In embodiments,alkoxysilyl acrylate has the formula

wherein L¹ is substituted or unsubstituted alkylene or substituted orunsubstituted heteroalkylene as described herein. In embodiments,alkoxysilyl methylacrylamide has the formula

wherein L¹ is substituted or unsubstituted alkylene or substituted orunsubstituted heteroalkylene as described herein. In embodiments,alkoxysilyl acrylamide has the formula

wherein L is substituted or unsubstituted alkylene or substituted orunsubstituted heteroalkylene as described herein.

The term “alkoxy” refers to an alkyl group bonded to an oxygen atom.

The term “nucleophile” as used herein refers to a chemical group that iscapable of donating electron density. All molecules or ions with a freepair of electrons or at least one pi bond can act as nucleophiles. Theterm “electrophile” as used herein refers to a chemical group that iscapable of accepting electron density. An “electrophilic substituent”,“electrophilic chemical moiety”, or “electrophilic moiety” refers to anelectron-poor chemical group, substituent, or moiety (monovalentchemical group), which may react with an electron-donating group, suchas a nucleophile, by accepting an electron pair or electron density toform a bond.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

II. Compositions & Kits

In an aspect is provided a solid support including two or more wells,wherein each well includes one or more particles as described herein. Inembodiments, each particle includes a plurality of oligonucleotidemoieties covalently attached to said particle via a polymericbioconjugate linker. In embodiments, the polymeric bioconjugate linkeris formed through a reaction between a particle polymer (e.g., a polymercovalently attached to the surface of the particle) including a firstbioconjugate reactive moiety and an oligonucleotide including a secondbioconjugate reactive moiety. In embodiments, the average longestdimension of the particle is from about 100 nm to about 1000 nm. Inembodiments, each particle includes a plurality of oligonucleotidemoieties covalently attached to said particle via a bioconjugate linker,wherein the polymeric bioconjugate linker is formed through a reactionbetween a particle polymer (e.g., a polymer covalently attached to thesurface of the particle) including a first bioconjugate reactive moiety(e.g., an azide) and an oligonucleotide including a second bioconjugatereactive moiety (e.g., DBCO).

In an aspect is provided an array (e.g., a multiwell container),including: a solid support including a surface, the surface comprising aplurality of wells separated from each other by interstitial regions onthe surface, wherein one or more wells includes a particle, wherein theparticle includes a plurality of bioconjugate reactive moieties, aplurality of oligonucleotide moieties (e.g., covalently bound to theparticle), or a combination thereof. In embodiments, there is at leastone particle per well. In embodiments, there is at most one particle perwell. In embodiments, the surface is substantially free ofoligonucleotides. In embodiments, the surface does not includeoligonucleotide capture moieties.

In another aspect is provided an array, including: a solid supportincluding a surface, the surface including a plurality of wellsseparated from each other by interstitial regions on the surface,wherein the surface comprises a polymer layer and is substantially freeof oligonucleotide moieties, wherein one or more wells contains aparticle, wherein the particle includes a plurality of bioconjugatereactive moieties, a plurality of oligonucleotide moieties, or acombination thereof. In embodiments, there is at least one particle perwell. In embodiments, there two or more particles per well. Inembodiments, there is at most one particle per well. In embodiments, thesurface is substantially free of oligonucleotides. In embodiments, thesurface does not include oligonucleotide capture moieties.

In an aspect is provided a solid support (e.g., a patterned glass slideor planar support) including two or more wells, wherein each wellincludes a particle including a plurality of bioconjugate reactivemoieties, a plurality of oligonucleotide moieties, or a combinationthereof, wherein the average longest dimension of the particle is fromabout 100 nm to about 1000 nm. In embodiments, the solid supportincludes a plurality of wells (e.g., a billion or more wells). Inembodiments, the wells (e.g., each well) is separated by about 0.1 μm toabout 5.0 μm. In embodiments, the wells (e.g., each well) is separatedby about 0.2 μm to about 2.0 μm. In embodiments, the wells (e.g., eachwell) is separated by about 0.5 μm to about 1.5 μm. In embodiments, thewells of the solid support are all the same size. In embodiments, one ormore wells are different sizes (e.g., one population of wells are 1.0 μmin diameter, and a second population are 0.5 μm in diameter). Inembodiments, the solid support is a glass slide about 75 mm by about 25mm. In embodiments, the solid support includes a resist (e.g., aphotoresist or nanoimprint resist including a crosslinked polymer matrixattached to the solid support).

In an aspect is provided a solid support including two or more wells(alternatively referred to as a multiwell container or array). Inembodiments, wherein each well is separated by about 0.2 μm to about 2.0μm and each well includes at least one particle as described herein. Inembodiments, the particle (e.g., a nanoparticle) includes a plurality ofoligonucleotide moieties covalently attached to the particle via abioconjugate linker, wherein the bioconjugate linker is formed via areaction between a particle polymer including a first bioconjugatereactive moiety and an oligonucleotide including a second bioconjugatereactive moiety. In embodiments, the average longest dimension of theparticle is from about 100 nm to about 1000 nm. In embodiments, theparticle includes a plurality of particle polymers (e.g., a polymer orcopolymer described herein). In embodiments, the particle polymer is abrush polymer. In embodiments, the plurality of particle polymers arenot crosslinked (i.e., covalently bound to each other). In embodiments,the plurality of particle polymers are crosslinked.

In embodiments, density of wells on the solid support may be tuned. Forexample, in embodiments, the multiwell container includes a density ofat least about 100 wells per mm² about 1,000 wells per mm², about 0.1million wells per mm², about 1 million wells per mm² about 2 millionwells per mm², about 5 million wells per mm², about 10 million wells permm², about 50 million wells per mm², or more. In embodiments, themultiwell container includes no more than about 50 million wells permm², about 10 million wells per mm², about 5 million wells per mm²,about 2 million wells per mm², about 1 million wells per mm², about 0.1million wells per mm², about 1,000 wells per mm², about 100 wells permm², or less. In embodiments, the solid support includes about 500,1,000, 2,500, 5,000, or about 25,000 wells per mm². In embodiments, thesolid support includes about 1×10⁶ to about 1×10¹² wells. Inembodiments, the solid support includes about 1×10⁷ to about 1×10¹²wells. In embodiments, the solid support includes about 1×10⁸ to about1×10¹² wells. In embodiments, the solid support includes about 1×10⁶ toabout 1×10⁹ wells. In embodiments, the solid support includes about1×10⁹ to about 1×10¹⁰ wells. In embodiments, the solid support includesabout 1×10⁷ to about 1×10⁹ wells. In embodiments, the solid supportincludes about 1×10⁸ to about 1×10⁸ wells. In embodiments, the solidsupport includes about 1×10⁶ to about 1×10⁸ wells. In embodiments, thesolid support includes about 1×10⁶, 1×10⁷, 1×10⁸, 1×10⁹, 1×10¹⁰, 1×10¹¹,1×10¹², 5×10¹², or more wells. In embodiments, the solid supportincludes about 1.8×10⁹, 3.7×10⁹, 9.4×10⁹, 1.9×10¹⁰, or about 9.4×10¹⁰wells. In embodiments, the solid support includes about 1×10⁶ or morewells. In embodiments, the solid support includes about 1×10⁷ or morewells. In embodiments, the solid support includes about 1×10⁸ or morewells. In embodiments, the solid support includes about 1×10⁹ or morewells. In embodiments, the solid support includes about 1×10¹⁰ or morewells. In embodiments, the solid support includes about 1×10¹¹ or morewells. In embodiments, the solid support includes about 1×10¹² or morewells. In embodiments, the solid support is a glass slide. Inembodiments, the solid support is a about 75 mm by about 25 mm. Inembodiments, the solid support includes one, two, three, or fourchannels.

In embodiments, the solid support includes a polymer layer. Inembodiments, the polymer layer includes polymerized units of alkoxysilylmethacrylate, alkoxysilyl acrylate, alkoxysilyl methylacrylamide,alkoxysilyl methylacrylamide, or a copolymer thereof. In embodiments,the polymer layer includes polymerized units of alkoxysilylmethacrylate. In embodiments, the polymer layer includes polymerizedunits of alkoxysilyl acrylate. In embodiments, the polymer layerincludes polymerized units of alkoxysilyl methylacrylamide. Inembodiments, the polymer layer includes polymerized units of alkoxysilylmethylacrylamide. In embodiments, the polymer layer includesglycidyloxypropyl-trimethyloxysilane. In embodiments, the polymer layerincludes methacryloxypropyl-trimethoxysilane. In embodiments, thepolymer layer includes polymerized units of

or a copolymer thereof.

In embodiments, the solid support includes a photoresist, alternativelyreferred to herein as a resist. A “resist” as used herein is used inaccordance with its ordinary meaning in the art of lithography andrefers to a polymer matrix (e.g., a polymer network). In embodiments,the photoresist is a silsesquioxane resist, an epoxy-based polymerresist, poly(vinylpyrrolidone-vinyl acrylic acid) copolymer resist, anOff-stoichiometry thiol-enes (OSTE) resist, amorphous fluoropolymerresist, a crystalline fluoropolymer resist, polysiloxane resist, or aorganically modified ceramic polymer resist. In embodiments, thephotoresist is a silsesquioxane resist. In embodiments, the photoresistis an epoxy-based polymer resist. In embodiments, the photoresist is apoly(vinylpyrrolidone-vinyl acrylic acid) copolymer resist. Inembodiments, the photoresist is an Off-stoichiometry thiol-enes (OSTE)resist. In embodiments, the photoresist is an amorphous fluoropolymerresist. In embodiments, the photoresist is a crystalline fluoropolymerresist. In embodiments, the photoresist is a polysiloxane resist. Inembodiments, the photoresist is an organically modified ceramic polymerresist. In embodiments, the photoresist includes polymerized alkoxysilylmethacrylate polymers and metal oxides (e.g., SiO₂, ZrO, MgO, Al₂O₃,TiO₂ or Ta₂O₅). In embodiments, the photoresist includes polymerizedalkoxysilyl acrylate polymers and metal oxides (e.g., SiO₂, ZrO, MgO,Al₂O₃, TiO₂ or Ta₂O₅). In embodiments, the photoresist includes metalatoms, such as Si, Zr, Mg, Al, Ti or Ta atoms.

In embodiments, the wells are separated from each other by interstitialregions including a polymer layer as described herein (e.g., anamphiphilic copolymer). In embodiments, the solid support furtherincludes a photoresist, wherein the photoresist does not contact thebottom of the well. In embodiments, the polymer layer is substantiallyfree of oligonucleotide moieties. In embodiments, the solid support doesnot include a polymer (e.g., the solid support is a patterned glassslide). In embodiments, the wells do not include a polymer (e.g., anamphiphilic polymer as described herein) prior to particle loading. Inembodiments, the solid support further includes a photoresist, whereinthe photoresist is in contact the bottom of the well and theinterstitial space. In embodiments, the polymer layer is substantiallyfree of oligonucleotide moieties (e.g., oligonucleotide moieties are notcovalently bound to the solid support and/or the polymer layer,including the interstitial space). In embodiments, the wells include apolymer (e.g., an amphiphilic polymer and/or resist as described herein)prior to particle loading.

In embodiments, each well contains a plurality of particles (e.g.,wherein the well is at least twice the diameter of the longest dimensionof the particle). In embodiments, each well is referred to as a feature.In embodiments, the arrays include about 10,000,000 features/cm² toabout 5,000,000,000 features/cm². In embodiments, the arrays includeabout 100,000,000 features/cm² to about 1,000,000,000 features/cm². Inembodiments, the arrays include about 100,000 features/cm² to about100,000,000 features/cm². In embodiments, the arrays include about orabout 10,000,000 features/cm² to about 50,000,000 features/cm². Inembodiments, each well includes one particle. In embodiments, each wellinclude 10 to 100 nanoparticles (e.g., the diameter of the well is 5mm).

In embodiments, the wells have a mean or median separation from oneanother of about 0.5-5 μm. In embodiments, the mean or median separationis about 0.1-10 microns, 0.25-5 microns, 0.5-2 microns, 1 micron, or anumber or a range between any two of these values. In embodiments, themean or median separation is about or at least about 0.1, 0.2, 0.3, 0.4,0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8,1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2,3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6,4.7, 4.8, 4.9, 5.0 μm, or a number or a range between any two of thesevalues. In embodiments, the mean or median separation is about or atleast about 0.1 μm. In embodiments, the mean or median separation isabout or at least about 0.2 μm. In embodiments, the mean or medianseparation is about or at least about 0.3 μm. In embodiments, the meanor median separation is about or at least about 0.4 μm. In embodiments,the mean or median separation is about or at least about 0.5 μm. Inembodiments, the mean or median separation is about or at least about0.6 μm. In embodiments, the mean or median separation is about or atleast about 0.7 μm. In embodiments, the mean or median separation isabout or at least about 0.8 μm. In embodiments, the mean or medianseparation is about or at least about 0.9 μm. In embodiments, the meanor median separation is about or at least about 1.0 μm. In embodiments,the mean or median separation is about or at least about 1.1 μm. Inembodiments, the mean or median separation is about or at least about1.2 μm. In embodiments, the mean or median separation is about or atleast about 1.3 μm. In embodiments, the mean or median separation isabout or at least about 1.4 μm. In embodiments, the mean or medianseparation is about or at least about 1.5 μm. In embodiments, the meanor median separation is about or at least about 1.6 μm. In embodiments,the mean or median separation is about or at least about 1.7 μm. Inembodiments, the mean or median separation is about or at least about1.8 μm. In embodiments, the mean or median separation is about or atleast about 1.9 μm. In embodiments, the mean or median separation isabout or at least about 2.0 μm. In embodiments, the mean or medianseparation is about or at least about 2.1 μm. In embodiments, the meanor median separation is about or at least about 2.2 μm. In embodiments,the mean or median separation is about or at least about 2.3 μm. Inembodiments, the mean or median separation is about or at least about2.4 μm. In embodiments, the mean or median separation is about or atleast about 2.5 μm. In embodiments, the mean or median separation isabout or at least about 2.6 μm. In embodiments, the mean or medianseparation is about or at least about 2.7 μm. In embodiments, the meanor median separation is about or at least about 2.8 μm. In embodiments,the mean or median separation is about or at least about 2.9 μm. Inembodiments, the mean or median separation is about or at least about3.0 μm. In embodiments, the mean or median separation is about or atleast about 3.1 μm. In embodiments, the mean or median separation isabout or at least about 3.2 μm. In embodiments, the mean or medianseparation is about or at least about 3.3 μm. In embodiments, the meanor median separation is about or at least about 3.4 μm. In embodiments,the mean or median separation is about or at least about 3.5 μm. Inembodiments, the mean or median separation is about or at least about3.6 μm. In embodiments, the mean or median separation is about or atleast about 3.7 μm. In embodiments, the mean or median separation isabout or at least about 3.8 μm. In embodiments, the mean or medianseparation is about or at least about 3.9 μm. In embodiments, the meanor median separation is about or at least about 4.0 μm. In embodiments,the mean or median separation is about or at least about 4.1 μm. Inembodiments, the mean or median separation is about or at least about4.2 μm. In embodiments, the mean or median separation is about or atleast about 4.3 μm. In embodiments, the mean or median separation isabout or at least about 4.4 μm. In embodiments, the mean or medianseparation is about or at least about 4.5 μm. In embodiments, the meanor median separation is about or at least about 4.6 μm. In embodiments,the mean or median separation is about or at least about 4.7 μm. Inembodiments, the mean or median separation is about or at least about4.8 μm. In embodiments, the mean or median separation is about or atleast about 4.9 μm. In embodiments, the mean or median separation isabout or at least about 5.0 μm. The mean or median separation may bemeasured center-to-center (i.e., the center of one well to the center ofa second well). In embodiments of the methods provided herein, the wellshave a mean or median separation (measured center-to-center) from oneanother of about 0.5-5 μm. The mean or median separation may be measurededge-to-edge (i.e., the edge of well to the edge of a second well). Inembodiments, the wells have a mean or median separation (measurededge-to-edge) from one another of about 0.2-1.5 μm. In embodiments, thewells have a mean or median separation (measured center-to-center) fromone another of about 0.7-1.5 μm.

Neighboring features of an array can be discrete one from the other inthat they do not overlap. Accordingly, the features can be adjacent toeach other or separated by a gap (e.g., an interstitial space). Inembodiments where features are spaced apart, neighboring sites can beseparated, for example, by a distance of less than 10 μm, 5 μm, 1 μm,0.9 μm, 0.8 μm, 0.7 μm, 0.6 μm, 0.5 μm, or less. The layout of featureson an array can also be understood in terms of center-to-centerdistances between neighboring features. An array useful in the inventioncan have neighboring features with center-to-center spacing of less thanabout 10 μm, 5 μm, 1 μm, 0.9 μm, 0.8 μm, 0.7 μm, 0.6 μm, 0.5 μm, 0.4 μm,or less. In embodiments, the array has neighboring features withcenter-to-center spacing of less than about 10 μm. In embodiments, thearray has neighboring features with center-to-center spacing of lessthan about 5 μm. In embodiments, the array has neighboring features withcenter-to-center spacing of less than about 1 μm. In embodiments, thearray has neighboring features with center-to-center spacing of lessthan about 0.9 μm. In embodiments, the array has neighboring featureswith center-to-center spacing of less than about 0.8 μm. In embodiments,the array has neighboring features with center-to-center spacing of lessthan about 0.7 μm. In embodiments, the array has neighboring featureswith center-to-center spacing of less than about 0.6 μm. In embodiments,the array has neighboring features with center-to-center spacing of lessthan about 0.5 μm. In embodiments, the array has neighboring featureswith center-to-center spacing of less than about 0.4 μm. Furthermore, itwill be understood that the distance values described above andelsewhere herein can represent an average distance between neighboringfeatures of an array. As such, not all neighboring features need to fallin the specified range unless specifically indicated to the contrary,for example, by a specific statement that the distance constitutes athreshold distance between all neighboring features of an array.

The arrays and solid supports for some embodiments have at least onesurface located within a flow cell. Flow cells provide a convenientformat for housing an array of clusters produced by the methodsdescribed herein, in particular when subjected to an SBS or otherdetection technique that involves repeated delivery of reagents incycles.

In some embodiments, the particle is a functionalized particle includinga particle core and a particle shell, wherein said particle shellincludes the plurality of bioconjugate reactive moieties, the pluralityof oligonucleotide moieties, or a combination thereof, wherein each ofthe bioconjugate reactive moieties and each of the oligonucleotidemoieties includes a linker binding the bioconjugate reactive moietiesand oligonucleotide to the particle core.

In embodiments, the particle is a functionalized particle including aparticle core (e.g., a silica core) and a plurality of polymer moieties,wherein each polymer moiety includes a plurality of bioconjugatereactive moieties, a plurality of oligonucleotide moieties (e.g.,oligonucleotide moieties covalently bound to the particle polymer), or acombination thereof, wherein each of the bioconjugate reactive moietiesand each of the oligonucleotide moieties includes a linker binding thebioconjugate reactive moieties and oligonucleotide to the particle core.In embodiments, the particle includes a particle core and a particlepolymer, wherein the bioconjugate reactive moieties and oligonucleotidemoieties are covalently attached via a linker to the particle polymer.For example, FIGS. 10A-10B illustrate an embodiment of a functionalizedparticle as described herein. The particle polymer, as illustrated inFIGS. 10A-10B, includes a polymer (e.g., PEGMA) linked with bioconjugatereactive moieties (e.g., GMA-Az).

In some embodiments, the particle core includes glass, ceramic, metal,silica, magnetic material, or a paramagnetic material. The particle coremay be an inorganic particle core. The inorganic particle core may be ametal particle core. When the particle core is a metal, the metal may betitanium, zirconium, gold, silver, platinum, cerium, arsenic, iron,aluminum or silicon. The metal particle core may be titanium, zirconium,gold, silver, or platinum and appropriate metal oxides thereof. Inembodiments, the particle core is titanium oxide, zirconium oxide,cerium oxide, arsenic oxide, iron oxide, aluminum oxide, or siliconoxide. The metal oxide particle core may be titanium oxide or zirconiumoxide. The particle may be titanium. The particle may be gold. Theparticle may be silicon dioxide. The particle may be silica. Inembodiments, the particle core is in the form of a bead. For example,the core/shell layers may be formed around a supporting structure, forexample, a silica, magnetic, or paramagnetic bead. In some embodiments,the composition includes a solid bead support (which itself may includea magnetic core and an encapsulating polymer layer), a functional corelayer around the bead for primer attachment, and a shell polymer layerin which no amplification reactions take place. In embodiments, theparticle is a silica particle includes a magnetic core, and a copolymershell. In embodiments, the particle shell is chemically distinct fromthe particle core.

In embodiments, the particle core includes glass, ceramic, metal,silica, magnetic material, or a paramagnetic material. The particle coremay be an inorganic particle core. The inorganic particle core may be ametal particle core. When the particle core is a metal, the metal may betitanium, zirconium, gold, silver, platinum, cerium, arsenic, iron,aluminum or silicon. The metal particle core may be titanium, zirconium,gold, silver, or platinum and appropriate metal oxides thereof. Inembodiments, the particle core is titanium oxide, zirconium oxide,cerium oxide, arsenic oxide, iron oxide, aluminum oxide, or siliconoxide. The metal oxide particle core may be titanium oxide or zirconiumoxide. The particle may be titanium. The particle may be gold. Theparticle may be silicon dioxide. The particle may be silica. Inembodiments, the particle core is in the form of a bead. For example,the core/shell layers may be formed around a supporting structure, forexample, a silica, magnetic, or paramagnetic bead. In embodiments, thecomposition includes a solid bead support (which itself may include amagnetic core and an encapsulating polymer layer), a functional corelayer around the bead for primer attachment. In embodiments, theparticle is a silica particle further includes a magnetic core. Inembodiments, the particle polymer is chemically distinct from theparticle core. In embodiments, the particle polymer is permeable toamplification and/or sequencing reagents (e.g., a polymerase,nucleotides, salts, and buffers). In embodiments, the particle includesglass or silica. In embodiments, the particle is a silica nanoparticle.

In some embodiments, the particle shell includes polymerized units ofpolyacrylamide (AAm), poly-N-isopropylacrylamide, polyN-isopropylpolyacrylamide, sulfobetaine acrylate (SBA), carboxybetaineacrylate (CBA), phosphorylcholine acrylate (PCA), sulfobetainemethacrylate (SBMA), carboxybetaine methacrylate (CBMA),phosphorylcholine methacrylate (PCMA), polyethylene glycol acrylate,methacrylate, polyethylene glycol (PEG)-thiol/PEG-acrylate,acrylamide/N,N′-bis(acryloyl)cystamine (BACy), PEG/polypropylene oxide(PPO), polyacrylic acid, poly(hydroxyethyl methacrylate) (PHEMA),poly(methyl methacrylate) (PMMA), poly(N-isopropylacrylamide) (PNIPAAm),poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA),polycaprolactone (PCL), poly(vinylsulfonic acid) (PVSA), poly(L-asparticacid), poly(L-glutamic acid), polylysine, agar, agarose, alginate,heparin, alginate sulfate, dextran sulfate, hyaluronan, pectin,carrageenan, gelatin, chitosan, cellulose, collagen, glicydylmethacrylate (GMA), hydroxyethylmethacrylate (HEMA),hydroxyethylacrylate (HEA), hydroxypropylmethacrylate (HPMA),polyethylene glycol methacrylate (PEGMA), polyethylene glycol acrylate(PEGA), isocyanatoethyl methacrylate (IEM), or a copolymer thereof. Inembodiments, the particle shell includes polymerized units ofpolyacrylamide (AAm), glicydyl methacrylate (GMA), polyethylene glycolmethacrylate (PEGMA), polyethylene glycol methacrylate (PEGMA),isocyanatoethyl methacrylate (IEM), or a copolymer thereof. Inembodiments, the particle shell includes polymerized units ofpolyethylene glycol methacrylate (PEGMA) and glicydyl methacrylate(GMA). In embodiments, the particle shell includes polymerized units ofpolyethylene glycol methacrylate (PEGMA) and isocyanatoethylmethacrylate (IEM). In embodiments, the particle shell includespolymerized units of 3-azido-2-hydroxypropyl methacrylate,2-azido-3-hydroxypropyl methacrylate,2-(((2-azidoethoxy)carbonyl)amino)ethyl methacrylate,3-azido-2-hydroxypropyl acrylate, 2-azido-3-hydroxypropyl acrylate, or2-(((2-azidoethoxy)carbonyl)amino)ethyl acrylate. In embodiments, theparticle shell includes polymerized units of 3-azido-2-hydroxypropylmethacrylate, 2-azido-3-hydroxypropyl methacrylate, or2-(((2-azidoethoxy)carbonyl)amino)ethyl methacrylate. In embodiments,the particle shell includes polymerized units of 3-azido-2-hydroxypropylmethacrylate. In embodiments, the particle shell includes polymerizedunits of 3-azido-2-hydroxypropyl methacrylate 2-azido-3-hydroxypropylmethacrylate. In embodiments, the particle shell includes polymerizedunits of 3-azido-2-hydroxypropyl methacrylate2-(((2-azidoethoxy)carbonyl)amino)ethyl methacrylate.

In some embodiments, the particle polymer includes polymerized units ofpolyacrylamide (AAm), poly-N-isopropylacrylamide, polyN-isopropylpolyacrylamide, sulfobetaine acrylate (SBA), carboxybetaineacrylate (CBA), phosphorylcholine acrylate (PCA), sulfobetainemethacrylate (SBMA), carboxybetaine methacrylate (CBMA),phosphorylcholine methacrylate (PCMA), polyethylene glycol acrylate,methacrylate, N-vinyl pyrrolidone, polyethylene glycol(PEG)-thiol/PEG-acrylate, acrylamide/N,N′-bis(acryloyl)cystamine (BACy),PEG/polypropylene oxide (PPO), polyacrylic acid, poly(hydroxyethylmethacrylate) (PHEMA), poly(methyl methacrylate) (PMMA),poly(N-isopropylacrylamide) (PNIPAAm), poly(lactic acid) (PLA),poly(lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL),poly(vinylsulfonic acid) (PVSA), poly(L-aspartic acid), poly(L-glutamicacid), polylysine, agar, agarose, alginate, heparin, alginate sulfate,dextran sulfate, hyaluronan, pectin, carrageenan, gelatin, chitosan,cellulose, collagen, glicydyl methacrylate (GMA), glicydyl methacrylate(GMA) azide, hydroxyethylmethacrylate (HEMA), hydroxyethylacrylate(HEA), hydroxypropylmethacrylate (HPMA), polyethylene glycolmethacrylate (PEGMA), polyethylene glycol acrylate (PEGA),isocyanatoethyl methacrylate (IEM), or a copolymer thereof. Inembodiments, the particle polymer includes polymerized units ofpolyethylene glycol methacrylate (PEGMA) and glicydyl methacrylate(GMA). In embodiments, the particle polymer includes polymerized unitsof polyethylene glycol methacrylate (PEGMA) and isocyanatoethylmethacrylate (IEM). In embodiments, the particle polymer includespolymerized units of glicydyl methacrylate azide (GMA azide) andpolyethylene glycol methacrylate (PEGMA). In embodiments, the ratio ofGMA azide to PEGMA is 1:1. In embodiments, the ratio of GMA azide toPEGMA is 1:2. In embodiments, the ratio of GMA azide to PEGMA is 1:3. Inembodiments, the ratio of GMA azide to PEGMA is 1:4. In embodiments, theratio of GMA azide to PEGMA is 1:5. In embodiments, the ratio of GMAazide to PEGMA is 1:6. In embodiments, the ratio of GMA azide to PEGMAis 1:7. In embodiments, the ratio of GMA azide to PEGMA is 1:8. Inembodiments, the particle polymer includes polymerized units of3-azido-2-hydroxypropyl methacrylate, 2-azido-3-hydroxypropylmethacrylate, 2-(((2-azidoethoxy)carbonyl)amino)ethyl methacrylate,3-azido-2-hydroxypropyl acrylate, 2-azido-3-hydroxypropyl acrylate, or2-(((2-azidoethoxy)carbonyl)amino)ethyl acrylate. In embodiments, theparticle polymer includes polymerized units of 3-azido-2-hydroxypropylmethacrylate, 2-azido-3-hydroxypropyl methacrylate, or2-(((2-azidoethoxy)carbonyl)amino)ethyl methacrylate. In embodiments,the particle polymer includes polymerized units of3-azido-2-hydroxypropyl methacrylate. In embodiments, the particlepolymer includes polymerized units of 3-azido-2-hydroxypropylmethacrylate 2-azido-3-hydroxypropyl methacrylate. In embodiments, theparticle polymer includes polymerized units of 3-azido-2-hydroxypropylmethacrylate 2-(((2-azidoethoxy)carbonyl)amino)ethyl methacrylate. Inembodiments, the particle polymer includes polymerized units of a)polyethylene glycol methacrylate (PEGMA) and glicydyl methacrylate(GMA), b) polyethylene glycol methacrylate (PEGMA) and isocyanatoethylmethacrylate (IEM), or c) polyethylene glycol methacrylate (PEGMA) andglicydyl methacrylate (GMA) azide, wherein the particle core is a silicaparticle. In embodiments, the particle includes a plurality of particlepolymers (e.g., a plurality of polyethylene glycol methacrylate (PEGMA)and glicydyl methacrylate (GMA) azide copolymers). In embodiments, theparticle includes a plurality of brush particle polymers.

In embodiments, the particle polymer is covalently bound to theparticle. For example, the particle polymer is attached to the particlevia a polymerization initiator, for example (3-trimethoxysilyl)propyl2-bromo-2-methylpropionate, wherein the —Si—O— moieties are attached toa silica particle. In embodiments, the polymerization initiator has theformula:

In some embodiments, the particle is a polymer particle includingpolymerized units of polyacrylamide (AAm), poly-N-isopropylacrylamide,poly N-isopropylpolyacrylamide, sulfobetaine acrylate (SBA),carboxybetaine acrylate (CBA), phosphorylcholine acrylate (PCA),sulfobetaine methacrylate (SBMA), carboxybetaine methacrylate (CBMA),phosphorylcholine methacrylate (PCMA), polyethylene glycol acrylate,methacrylate, polyethylene glycol (PEG)-thiol/PEG-acrylate,acrylamide/N,N′-bis(acryloyl)cystamine (BACy), PEG/polypropylene oxide(PPO), polyacrylic acid, poly(hydroxyethyl methacrylate) (PHEMA),poly(methyl methacrylate) (PMMA), poly(N-isopropylacrylamide) (PNIPAAm),poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA),polycaprolactone (PCL), poly(vinylsulfonic acid) (PVSA), poly(L-asparticacid), poly(L-glutamic acid), polylysine, agar, agarose, alginate,heparin, alginate sulfate, dextran sulfate, hyaluronan, pectin,carrageenan, gelatin, chitosan, cellulose, collagen, glicydylmethacrylate (GMA), hydroxyethylmethacrylate (HEMA),hydroxyethylacrylate (HEA), hydroxypropylmethacrylate (HPMA),polyethylene glycol methacrylate (PEGMA), polyethylene glycol acrylate(PEGA), isocyanatoethyl methacrylate (IEM), or a copolymer thereof. Insome embodiments, the particle is a polymer particle includingpolymerized units of polyacrylamide (AAm), hydroxyethylmethacrylate(HEMA), hydroxyethylacrylate (HEA), hydroxypropylmethacrylate (HPMA),polyethylene glycol (PEG), isocyanatoethyl methacrylate (IEM), or acopolymer thereof. In embodiments, the particle is a polymer particleincluding polymerized units of polyacrylamide (AAm), glicydylmethacrylate (GMA), polyethylene glycol methacrylate (PEGMA),polyethylene glycol acrylate (PEGA), isocyanatoethyl methacrylate (IEM),or a copolymer thereof. In embodiments, the particle is a polymerparticle including polymerized units of polyacrylamide (AAm) andglicydyl methacrylate (GMA). In embodiments, the particle is a polymerparticle including polymerized units of polyacrylamide (AAm) andisocyanatoethyl methacrylate (IEM). In embodiments, the particle is apolymer particle including polymerized units of glicydyl methacrylate(GMA). In embodiments, the particle is a polymer particle includingpolymerized units of 3-azido-2-hydroxypropyl methacrylate,2-azido-3-hydroxypropyl methacrylate,2-(((2-azidoethoxy)carbonyl)amino)ethyl methacrylate,3-azido-2-hydroxypropyl acrylate, 2-azido-3-hydroxypropyl acrylate, or2-(((2-azidoethoxy)carbonyl)amino)ethyl acrylate. In embodiments, theparticle is a polymer particle including polymerized units of3-azido-2-hydroxypropyl methacrylate. In embodiments, the particle is apolymer particle including polymerized units of 2-azido-3-hydroxypropylmethacrylate. In embodiments, the particle is a polymer particleincluding polymerized units of 2-(((2-azidoethoxy)carbonyl)amino)ethylmethacrylate. In embodiments, the particle is a polymer particleincluding polymerized units of 3-azido-2-hydroxypropyl acrylate. Inembodiments, the particle is a polymer particle including polymerizedunits of 2-azido-3-hydroxypropyl acrylate. In embodiments, the particleis a polymer particle including polymerized units of2-(((2-azidoethoxy)carbonyl)amino)ethyl acrylate.

In embodiments, the particle is a polymeric particle includingpolymerized units of polyacrylamide (AAm), poly-N-isopropylacrylamide,poly N-isopropylpolyacrylamide, sulfobetaine acrylate (SBA),carboxybetaine acrylate (CBA), phosphorylcholine acrylate (PCA),sulfobetaine methacrylate (SBMA), carboxybetaine methacrylate (CBMA),phosphorylcholine methacrylate (PCMA), polyethylene glycol acrylate,methacrylate, polyethylene glycol (PEG)-thiol/PEG-acrylate,acrylamide/N,N′-bis(acryloyl)cystamine (BACy), PEG/polypropylene oxide(PPO), polyacrylic acid, poly(hydroxyethyl methacrylate) (PHEMA),poly(methyl methacrylate) (PMMA), poly(N-isopropylacrylamide) (PNIPAAm),poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA),polycaprolactone (PCL), poly(vinylsulfonic acid) (PVSA), poly(L-asparticacid), poly(L-glutamic acid), polylysine, agar, agarose, alginate,heparin, alginate sulfate, dextran sulfate, hyaluronan, pectin,carrageenan, gelatin, chitosan, cellulose, collagen, glicydylmethacrylate (GMA), hydroxyethylmethacrylate (HEMA),hydroxyethylacrylate (HEA), hydroxypropylmethacrylate (HPMA),polyethylene glycol methacrylate (PEGMA), polyethylene glycol acrylate(PEGA), isocyanatoethyl methacrylate (IEM), or a copolymer thereof. Insome embodiments, the particle is a polymeric particle includingpolymerized units of polyacrylamide (AAm), hydroxyethylmethacrylate(HEMA), hydroxyethylacrylate (HEA), hydroxypropylmethacrylate (HPMA),polyethylene glycol (PEG), isocyanatoethyl methacrylate (IEM), or acopolymer thereof. In embodiments, the particle is a polymeric particleincluding polymerized units of polyacrylamide (AAm), glicydylmethacrylate (GMA), polyethylene glycol methacrylate (PEGMA),polyethylene glycol acrylate (PEGA), isocyanatoethyl methacrylate (IEM),or a copolymer thereof. In embodiments, the particle is a polymericparticle including polymerized units of polyacrylamide (AAm) andglicydyl methacrylate (GMA). In embodiments, the particle is a polymericparticle including polymerized units of polyacrylamide (AAm) andisocyanatoethyl methacrylate (IEM). In embodiments, the particle is apolymeric particle including polymerized units of glicydyl methacrylate(GMA). In embodiments, the particle is a polymeric particle includingpolymerized units of glicydyl methacrylate azide (GMA) azide. Inembodiments, the particle is a polymeric particle including polymerizedunits of 3-azido-2-hydroxypropyl methacrylate, 2-azido-3-hydroxypropylmethacrylate, 2-(((2-azidoethoxy)carbonyl)amino)ethyl methacrylate,3-azido-2-hydroxypropyl acrylate, 2-azido-3-hydroxypropyl acrylate, or2-(((2-azidoethoxy)carbonyl)amino)ethyl acrylate. In embodiments, theparticle is a polymeric particle including polymerized units of3-azido-2-hydroxypropyl methacrylate. In embodiments, the particle is apolymeric particle including polymerized units of2-azido-3-hydroxypropyl methacrylate. In embodiments, the particle is apolymeric particle including polymerized units of2-(((2-azidoethoxy)carbonyl)amino)ethyl methacrylate. In embodiments,the particle is a polymeric particle including polymerized units of3-azido-2-hydroxypropyl acrylate. In embodiments, the particle is apolymeric particle including polymerized units of2-azido-3-hydroxypropyl acrylate. In embodiments, the particle is apolymeric particle including polymerized units of2-(((2-azidoethoxy)carbonyl)amino)ethyl acrylate. In embodiments, thepolymeric particle is permeable to a polymerase.

The polymer may be polymerized from a mixture of functionalized andnon-functionalized monomers, such that at least some functionalizedmonomers that provide attachment points (e.g., azide moieties) forprimers (e.g., DBCO-containing oligonucleotide primers) are spaced fromone another by one or more monomers lacking such attachment points(e.g., PEG or AAm). The frequency of monomer units attached to primerswithin a polymer can be adjusted by changing the concentration of thecorresponding functionalized monomer in the mixture of monomers. Inembodiments, monomer units of the core polymer that are attached to apolynucleotide primer are separated by, on average, about or at leastabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, or moremonomer units that are not attached to a primer, referred to herein as(ng). In embodiments, monomer units of the core polymer that areattached to a polynucleotide primer (referred to herein asoligonucleotide moieties) are separated by, on average, about or atleast about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, ormore monomer units that are not attached to a primer, referred to hereinas (ng). In embodiments, monomer units of the core polymer that areattached to a polynucleotide primer are separated by, on average, aboutor at least about 4 to 8 monomer units that are not attached to aprimer. In embodiments, monomer units of the core polymer that areattached to a polynucleotide primer are separated by, on average, about4 to 8 monomer units that are not attached to a primer. In embodiments,monomer units of the core polymer that are attached to a polynucleotideprimer are separated by, on average, about or at least about 6, 7, or 8monomer units that are not attached to a primer. In embodiments,primer-attached monomers are separated by, on average, about 1-50, 2-40,3-30, 4-25, or 5-20 monomers not attached to primers. In embodiments,monomer units of the core polymer that are attached to a polynucleotideprimer are separated by 3 monomer units that are not attached to aprimer (aka 3 ng). In embodiments, monomer units of the core polymerthat are attached to a polynucleotide primer are separated by 6 ng. Inembodiments, monomer units the core polymer that are attached to apolynucleotide primer are separated by 9 ng. The mixture can includemonomers with different functional groups (e.g., azides, alkynes, DBCO,etc.) as described herein.

In embodiments, the particles are non-covalently attached to the wells.In embodiments, the particles are physiosorbed to the wells. Inembodiments, the particles are covalently attached to the wells. Inembodiments, each particle attaches to the polymer layer of the surface(e.g., non-covalently attach to the polymer layer). In embodiments, theparticles contact the well and remain attached without any additionalmeans for attachment (e.g., hybridization of complementaryoligonucleotides immobilized on the solid support). In embodiments, thesolid support does not include immobilized oligonucleotides.

The manner in which an oligonucleotide primer is attached to the polymerwill depend on the type of functional group used to form the attachment.A variety of suitable functional groups are available, examples of whichare provided herein. The polymer may be polymerized from a mixture offunctionalized and non-functionalized monomers, and/or a mixture ofmonomers with different functional groups. In embodiments, functionalgroups are selected that specifically react with their intended target(e.g., a paired functional group attached to a desired target, such as aprimer), while also exhibiting anti-fouling characteristics thatprevent, or have a reduced propensity for, non-specific binding ofenzymes, dye-labeled nucleotides, and nucleic acids.

In embodiments, the particle shell is permeable to a polymerase. Inembodiments, the polymer particle is permeable to a polymerase. Inembodiments, the particle shell is permeable to an amplificationreaction mixture and/or a sequencing reaction mixture. In embodiments,the polymer particle is permeable to an amplification reaction mixtureand/or a sequencing reaction mixture. In embodiments, the particle shellis permeable to a sequencing reaction mixture. In embodiments, thepolymer particle is permeable to a sequencing reaction mixture. Inembodiments, the polymer particle and shell polymer are permeable to apolymerase for amplifying the target polynucleotide. In embodiments, theshell polymer has a higher permeability than the core (e.g., theparticle core is substantially less permeable than the particle shell).In embodiments, the polymer shell is permeable to a polymerase foramplifying the target polynucleotide, such that the interface of thecore is in contact with the polymerase. The term “sequencing reactionmixture” refers to an aqueous mixture that contains the agents andreagents necessary to allow addition of a nucleotide to a polynucleotidestrand by a polymerase (e.g., addition of a dNTP or dNTP analogue to aDNA strand by a DNA polymerase). Exemplary mixtures of agents andreagents include buffers (e.g., saline-sodium citrate (SSC),tris(hydroxymethyl)aminomethane or “Tris”), salts (e.g., KCl or(NH₄)₂SO₄)), nucleotides (e.g., modified nucleotides), polymerases,cleaving agent (e.g., tri-n-butyl-phosphine, triphenyl phosphine and itssulfonated versions (i.e., tris(3-sulfophenyl)-phosphine, TPPTS), andtri(carboxyethyl)phosphine (TCEP) and its salts, cleaving agentscavenger compounds (e.g., 2′-Dithiobisethanamine or11-Azido-3,6,9-trioxaundecane-1-amine), detergents and/or crowdingagents (e.g., PEG, Tween, BSA). In embodiments, the modified nucleotidesare reversibly terminated nucleotides linked to fluorescent dyes, suchthat the identity of a nucleotide added in a sequencing reaction can beidentified based on the fluorescent dye with which it is associated. Theterm “amplification reaction mixture” refers to an aqueous mixture thatcontains the agents and reagents necessary to make one or more copies ofa nucleic acid. Exemplary components include s polymerase, a nucleicacid template, a suitable primer or set of primers, suitable nucleotides(e.g., dNTPs), and a suitable buffer.

In embodiments, the particle polymer is permeable to a polymerase. Inembodiments, the polymeric particle is permeable to a polymerase. Inembodiments, the particle polymer is permeable to an amplificationreaction mixture and/or a sequencing reaction mixture. In embodiments,the polymeric particle is permeable to an amplification reaction mixtureand/or a sequencing reaction mixture. In embodiments, the polymericparticle is permeable to a sequencing reaction mixture. The term“sequencing reaction mixture” refers to an aqueous mixture that containsthe agents and reagents necessary to allow addition of a nucleotide to apolynucleotide strand by a polymerase (e.g., addition of a dNTP or dNTPanalogue to a DNA strand by a DNA polymerase). Exemplary mixtures ofagents and reagents include buffers (e.g., saline-sodium citrate (SSC),tris(hydroxymethyl)aminomethane or “Tris” or TE), salts (e.g., KCl or(NH₄)₂SO₄)), nucleotides (e.g., modified nucleotides), polymerases,cleaving agent (e.g., tri-n-butyl-phosphine, triphenyl phosphine and itssulfonated versions (i.e., tris(3-sulfophenyl)-phosphine, TPPTS), andtri(carboxyethyl)phosphine (TCEP) and its salts, cleaving agentscavenger compounds (e.g., 2′-Dithiobisethanamine or11-Azido-3,6,9-trioxaundecane-1-amine), detergents and/or crowdingagents (e.g., PEG, Tween, BSA). In embodiments, the modified nucleotidesare reversibly terminated nucleotides linked to fluorescent dyes, suchthat the identity of a nucleotide added in a sequencing reaction can beidentified based on the fluorescent dye with which it is associated. Theterm “amplification reaction mixture” refers to an aqueous mixture thatcontains the agents and reagents necessary to make one or more copies ofa nucleic acid. Exemplary components includes a polymerase, a nucleicacid template, a suitable primer or set of primers, suitable nucleotides(e.g., dNTPs), and a suitable buffer.

In embodiments, the solid support is subjected to lithographicpatterning methods (e.g., nanolithographic to microlithographicpatterning). In embodiments, prior to contacting the solid support witha plurality of particles, the solid support is subjected to lithographicpatterning methods (e.g., nanolithographic to microlithographicpatterning). Typically, features smaller than 10 micrometers areconsidered microlithographic, and features smaller than 100 nanometersare considered nanolithographic. Lithographic techniques make use ofmasks or templates to transfer patterns over a large areasimultaneously. A powerful microfabrication technique isphotolithography, i.e. the lithography using a UV light source and aphotosensitive material as resist. As the name suggests, the photoresist(alternatively referred to as a resist) is an active material layer thatcan be patterned by selective exposure and must “resist”chemical/physical attach of the underlying substrate. In embodiments,the resist is a crosslinked polymer matrix. In embodiments, the resistincludes silsesquioxane molecules. In embodiments, the resist includespolymerized epoxy-containing monomers, or polymerizedpoly(vinylpyrrolidone-vinyl acrylic acid) copolymers. In embodiments,the solid support includes a glass substrate having a surface coated insilsesquioxane resist (e.g., polyhedral oligosilsesquioxanemethacrylate(POSS)), an epoxy-based polymer resist (e.g., SU-8 as described in U.S.Pat. No. 4,882,245), poly(vinylpyrrolidone-vinyl acrylic acid) copolymerresist (e.g., as described in U.S. Pat. No. 7,467,632), or novolaksresist, bisazides resist, or a combination thereof (e.g., as describedin U.S. Pat. No. 4,970,276). In embodiments, the resist is removed priorto loading. Alternatively, in embodiments, the resist includes theplurality of wells and remains in contact with the solid support whilecontacting the support with a plurality of particles.

In embodiments, the solid support includes a photoresist. A photoresistis a light-sensitive polymer material used to form a patterned coatingon a surface. The process begins by coating a substrate (e.g., a glasssubstrate) with a light-sensitive organic material. A mask with thedesired pattern is used to block light so that only unmasked regions ofthe material will be exposed to light. In the case of a positivephotoresist, the photo-sensitive material is degraded by light and asuitable solvent will dissolve away the regions that were exposed tolight, leaving behind a coating where the mask was placed. In the caseof a negative photoresist, the photosensitive material is strengthened(either polymerized or cross-linked) by light, and a suitable solventwill dissolve away only the regions that were not exposed to light,leaving behind a coating in areas where the mask was not placed. Inembodiments, the solid support includes an epoxy-based photoresist(e.g., SU-8, SU-8 2000, SU-8 3000, SU-8 GLM2060). In embodiments, thesolid support includes a negative photoresist. Negative refers to aphotoresist whereby the parts exposed to UV become cross-linked (i.e.,immobilized), while the remainder of the polymer remains soluble and canbe washed away during development. In embodiments, the solid supportincludes an Off-stoichiometry thiol-enes (OSTE) polymer (e.g., an OSTEresist). In embodiments, the solid support includes an HydrogenSilsesquioxane (HSQ) polymer (e.g., HSQ resist). In embodiments, thesolid support includes a crosslinked polymer matrix on the surface ofthe wells and the interstitial regions.

In embodiments, the solid support includes a nanoimprint resist. Inembodiments, the solid support includes a photoresist and polymer layer,wherein the photoresist is between the solid support and the polymerlayer (e.g., as depicted in FIGS. 1A-1C). In embodiments the photoresistis on the interstitial areas and not the surface of the wells. Suitablephotoresist compositions are known in the art, such as, for example thecompositions and resins described in U.S. Pat. Nos. 6,897,012;6,991,888; 4,882,245; 7,467,632; 4,970,276, each of which isincorporated herein by reference in their entirety. In embodiments, thesolid support includes a photoresist and polymer layer, wherein thephotoresist is covalently attached to the solid support and covalentlyattached to the polymer layer. In embodiments, the resist is anamorphous (non-crystalline) fluoropolymer (e.g., CYTOP® from Bellex), acrystalline fluoropolymer, or a fluoropolymer having both amorphous andcrystalline domains. In embodiments, the resist is a suitablepolysiloxane, such as polydimethylsiloxane (PDMS).

In embodiments, the solid support includes a resist (e.g., a nanoimprintlithography (NIL) resist). Nanoimprint resists can include thermalcurable materials (e.g., thermoplastic polymers), and/or UV-curablepolymers. In embodiments, the solid support is generated by pressing atransparent mold possessing the pattern of interest (e.g., the patternof wells) into photo-curable liquid film, followed by solidifying theliquid materials via a UV light irradiation. Typical UV-curable resistshave low viscosity, low surface tension, and suitable adhesion to theglass substrate. For example, the solid support surface, but not thesurface of the wells, is coated in an organically modified ceramicpolymer (ORMOCER®, registered trademark of Fraunhofer-Gesellschaft zurFörderung der angewandten Forschung e. V. in Germany). Organicallymodified ceramics contain organic side chains attached to an inorganicsiloxane backbone. Several ORMOCER® polymers are now provided undernames such as “Ormocore”, “Ormoclad” and “Ormocomp” by Micro ResistTechnology GmbH. In embodiments, the solid support includes a resist asdescribed in Haas et al Volume 351, Issues 1-2, 30 Aug. 1999, Pages198-203, US 2015/0079351A1, US 2008/0000373, or US 2010/0160478, each ofwhich is incorporated herein by reference. In embodiments, the solidsupport surface, and the surface of the wells, is coated in anorganically modified ceramic polymer (ORMOCER®, registered trademark ofFraunhofer-Gesellschaft zur Förderung der angewandten Forschung e. V. inGermany). In embodiments, the resist (e.g., the organically modifiedceramic polymer) is not removed prior to particle deposition. Inembodiments, the wells are within the resist polymer and not the solidsupport.

In embodiments, the solid support includes a polymer layer(alternatively referred to as a polymer coating). In embodiments, thesolid support comprises a polymer layer, wherein the polymer layerincludes an amphiphilic copolymer. The term “amphiphilic copolymer” isused in accordance with its ordinary meaning and refers to a copolymercomposed of polymerized hydrophilic (e.g., PEG monomers) and hydrophobicmonomers (e.g., alkoxysilyl or (poly(propylene oxide) monomers). Theterm “amphiphilic copolymer” is used in accordance with its ordinarymeaning and refers to a copolymer composed of polymerized hydrophilic(e.g., PEG monomers or HEMA monomers) and hydrophobic monomers (e.g.,alkoxysilyl or (poly(propylene oxide) monomers). Amphiphilic copolymerscan have both hydrophilic and hydrophobic properties. In embodiments,the polymer layer includes an amphiphilic acrylate copolymer oramphiphilic methacrylate copolymer.

In embodiments, the amphiphilic polymer includes a poloxamer. In someembodiments, the solid support includes a poloxamer layer. In someembodiments, the poloxamer is a polyoxyethylene-polyoxypropylenecopolymers. In some embodiments, the poloxamer is poloxamer 101,poloxamer 105, poloxamer 108, poloxamer 122, poloxamer 123, poloxamer124, poloxamer 181, poloxamer 182, poloxamer 183, poloxamer 184,poloxamer 185, poloxamer 188, poloxamer 212, poloxamer 215, poloxamer217, poloxamer 231, poloxamer 234, poloxamer 235, poloxamer 237,poloxamer 238, poloxamer 282, poloxamer 284, poloxamer 288, poloxamer331, poloxamer 333, poloxamer 334, poloxamer 335, poloxamer 338,poloxamer 401, poloxamer 402, poloxamer 403, and poloxamer 407. Inembodiments, the poloxamer is poloxamer 184, poloxamer 188, poloxamer338, or poloxamer 407 (also known as F127).

In embodiments, the solid support includes a polymer layer, wherein thepolymer layer includes a brush copolymer or a comb polymer. A combpolymer includes a main polymer chain with two or more three-way branchpoints and linear side chains. A brush polymer includes a main polymerchain with linear, unbranched side chains and where one or more of thebranch points has four-way functionality or larger. See FIG. 7A as anexample illustration of a brush and comb polymer structure. Inembodiments, the polymer layer does not include oligonucleotidemoieties. In embodiments, the polymer layer is substantially free ofoligonucleotides. In embodiments, the polymer layer does not includeoligonucleotide capture moieties. In embodiments, the polymer layerbinds to the resist of the array. In embodiments, nucleic acid contentis not transferred to the solid support, rather the oligonucleotidemoieties are localized to the particle.

In some embodiments, the solid support includes a hydrophobic polymerlayer. In embodiments, the solid support includes a perfluorinatedpolymer. In embodiments, the solid support includes a polyfluorinatedpolymer. In embodiments, the solid support includes polymerized units ofa fluorine-containing methacrylate (e.g.,CH₂═C(CH₃)COOC—(CF₃)₂CF₂CF₂CF₃). Non-limiting examples and syntheticprotocols of fluorine-containing methacrylate monomers may be found inZhang, D., (2018). Materials (Basel, Switzerland), 11(11), 2258 (2018),which is incorporated herein by reference. In embodiments, thefluorinated polymer is an amorphous (non-crystalline) fluoropolymer(e.g., CYTOP® from Bellex), a crystalline fluoropolymer, or afluoropolymer having both amorphous and crystalline domains.

In some embodiments, the solid support includes a hydrophilic polymerlayer. In some embodiments, the hydrophilic polymer is a silanefunctionalized polymer. In some embodiments, the silane functionalizedpolymer is a silane functionalized polyethylene glycol (Si-PEG) polymeror a silane functionalized poly(acrylamide) (Si-PAm). In embodiments,the polymer layer is a silane functionalized polymer. In someembodiments, the silane functionalized polymer is silane functionalizedpoly(acrylamide) (Si-PAm).

In embodiments, the polymer layer or the amphiphilic polymer includespolymerized units of alkoxysilyl polymers. In embodiments, the polymerlayer includes polymerized units of alkoxysilyl polymers (e.g., TMSPM)and polymerized units of polyethylene glycol methacrylate (PEGMA); seefor example an embodiment of such a copolymer in FIG. 7B. Inembodiments, the amphiphilic copolymer includes polymerized units ofalkoxysilyl polymers and polymerized units of polyethylene glycolmethacrylate (PEGMA), or polyethylene glycol acrylate (PEGA). Inembodiments, the amphiphilic copolymer includes polymerized units of3-(trimethoxysilyl)propyl methacrylate (TMSPM),3-(trimethoxysilyl)propyl methacrylate (TMSPA) and polymerized units ofpolyethylene glycol methacrylate (PEGMA), or polyethylene glycolacrylate (PEGA). In embodiments, the amphiphilic copolymer comprisespolymerized units of 3-(trimethoxysilyl)propyl methacrylate (TMSPM) andpolymerized units of polyethylene glycol methacrylate (PEGMA). Inembodiments, the amphiphilic copolymer comprises polymerized units of3-(trimethoxysilyl)propyl methacrylate (TMSPM), polymerized units ofpolyethylene glycol methacrylate (PEGMA) and polymerized units ofhydroxyethylmethacrylate (HEMA). In embodiments, the amphiphiliccopolymer comprises polymerized units of polyethylene glycolmethacrylate (PEGMA) and polymerized units of hydroxyethylmethacrylate(HEMA).

In embodiments, the polymer layer includes polymerized units ofalkoxysilyl polymers having the formula:

wherein R¹ is H or methyl; R², R³, and R⁴ are each independentlysubstituted or unsubstituted alkyl or substituted or unsubstitutedheteroalkyl, wherein at least one of R², R³, and R⁴ includes an alkoxybond to the Si atom; and L¹ is substituted or unsubstituted alkylene orsubstituted or unsubstituted heteroalkylene. In embodiments, the polymerlayer further includes polymerized units of polyethylene glycolmethacrylate (PEGMA), polyethylene glycol acrylate (PEGA), sulfobetaineacrylate (SBA), carboxybetaine acrylate (CBA), phosphorylcholineacrylate (PCA), sulfobetaine methacrylate (SBMA), carboxybetainemethacrylate (CBMA), or phosphorylcholine methacrylate (PCMA). Inembodiments, R¹ is H. In embodiments, R¹ is unsubstituted methyl. Inembodiments, the polymer layer is an organically-modified ceramicpolymer. In embodiments, the polymer includes polymerized monomers ofalkoxysilyl polymers, such as

In embodiments, R² is R^(2A)-substituted or unsubstituted alkyl (e.g.,C₁-C₂₀, C₁₀-C₂₀, C₁-C₈, C₁-C₆, or C₁-C₄), or R^(2A)-substituted orunsubstituted heteroalkyl (e.g., 2 to 20, 8 to 20, 2 to 10, 2 to 8, 2 to6, or 2 to 4 membered). In embodiments, R² is substituted orunsubstituted alkyl (e.g., C₁-C₂₀, C₁₀-C₂₀, C₁-C₈, C₁-C₆, or C₁-C₄), orsubstituted or unsubstituted heteroalkyl (e.g., 2 to 20, 8 to 20, 2 to10, 2 to 8, 2 to 6, or 2 to 4 membered).

In embodiments, R² is unsubstituted —O—C₁-C₆ or —O—C₁-C₄ alkyl. Inembodiments, R² is unsubstituted —O—C₁-C₄ alkyl. In embodiments, R² isunsubstituted —O—C₁-C₆ alkyl. In embodiments, R² is unsubstituted—O-methyl. In embodiments, R² is unsubstituted —O—C₂ alkyl. Inembodiments, R² is unsubstituted —O—C₃ alkyl. In embodiments, R² isunsubstituted —O—C₄ alkyl. In embodiments, R² is unsubstituted —O—C₅alkyl. In embodiments, R² is unsubstituted —O—C₆ alkyl. In embodiments,R² is R^(2A)-substituted —O—C₁-C₆ or —O—C₁-C₄ alkyl. In embodiments, R²is R^(2A)-substituted —O—C₁-C₄ alkyl. In embodiments, R² isR^(2A)-substituted —O—C₁-C₆ alkyl. In embodiments, R² isR^(2A)-substituted —O-methyl. In embodiments, R² is R^(2A)-substituted—O—C₂ alkyl. In embodiments, R² is R^(2A)-substituted —O—C₃ alkyl. Inembodiments, R² is R^(2A)-substituted —O—C₄ alkyl. In embodiments, R² isR^(2A)-substituted —O—C₅ alkyl. In embodiments, R² is R^(2A)-substituted—O—C₆ alkyl. In embodiments, R² is R^(2A)-substituted 2 to 10 memberedheteroalkyl. In embodiments, R² is R^(2A)-substituted 2 to 8 memberedheteroalkyl. In embodiments, R² is R^(2A)-substituted 2 to 6 memberedheteroalkyl. In embodiments, R² is R^(2A)-substituted 2 to 4 memberedheteroalkyl. In embodiments, R² is an unsubstituted 2 to 10 memberedheteroalkyl. In embodiments, R² is an unsubstituted 2 to 8 memberedheteroalkyl. In embodiments, R² is an unsubstituted 2 to 6 memberedheteroalkyl. In embodiments, R² is an unsubstituted 2 to 4 memberedheteroalkyl.

R^(2A) is independently oxo, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CHCl₂,—CHBr₂, —CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I, —CN, —OH, —NH₂,—COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NH₄NH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃,—OCF₃, —OCBr₃, —OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl,—OCH₂Br, —OCH₂I, —OCH₂F, —N₃, —SF₅, —NH₃ ⁺, —SO₃ ⁻, —OPO₃H⁻, —SCN,—ONO₂, unsubstituted alkyl (e.g., C₁-C₂₀, C₁₀-C₂₀, C₁-C₈, C₁-C₆, orC₁-C₄), unsubstituted heteroalkyl (e.g., 2 to 20, 8 to 20, 2 to 10, 2 to8, 2 to 6, or 2 to 4 membered), unsubstituted cycloalkyl (e.g., C₃-C₈,C₃-C₆, or C₅-C₆), unsubstituted heterocycloalkyl (e.g., 3 to 8, 3 to 6,or 5 to 6 membered), unsubstituted aryl (e.g., C₆-C₁₀, C₁₀, or phenyl),or unsubstituted heteroaryl (e.g., 5 to 10, 5 to 9, or 5 to 6 membered).In embodiments, R^(2A) is —OH.

In embodiments, R³ is R^(3A)-substituted or unsubstituted alkyl (e.g.,C₁-C₂₀, C₁₀-C₂₀, C₁-C₈, C₁-C₆, or C₁-C₄), or R^(3A)-substituted orunsubstituted heteroalkyl (e.g., 2 to 20, 8 to 20, 2 to 10, 2 to 8, 2 to6, or 2 to 4 membered). In embodiments, R³ is substituted orunsubstituted alkyl (e.g., C₁-C₂₀, C₁₀-C₂₀, C₁-C₈, C₁-C₆, or C₁-C₄), orsubstituted or unsubstituted heteroalkyl (e.g., 2 to 20, 8 to 20, 2 to10, 2 to 8, 2 to 6, or 2 to 4 membered).

In embodiments, R³ is unsubstituted —O—C₁-C₆ or —O—C₁-C₄ alkyl. Inembodiments, R³ is unsubstituted —O—C₁-C₄ alkyl. In embodiments, R³ isunsubstituted —O—C₁-C₆ alkyl. In embodiments, R³ is unsubstituted—O-methyl. In embodiments, R³ is unsubstituted —O—C₂ alkyl. Inembodiments, R³ is unsubstituted —O—C₃ alkyl. In embodiments, R³ isunsubstituted —O—C₄ alkyl. In embodiments, R³ is unsubstituted —O—C₅alkyl. In embodiments, R³ is unsubstituted —O—C₆ alkyl. In embodiments,R³ is R^(3A)-substituted —O—C₁-C₆ or —O—C₁-C₄ alkyl. In embodiments, R³is R^(3A)-substituted —O—C₁-C₄ alkyl. In embodiments, R³ isR^(3A)-substituted —O—C₁-C₆ alkyl. In embodiments, R³ isR^(3A)-substituted —O-methyl. In embodiments, R³ is R^(3A)-substituted—O—C₂ alkyl. In embodiments, R³ is R^(3A)-substituted —O—C₃ alkyl. Inembodiments, R³ is R^(3A)-substituted —O—C₄ alkyl. In embodiments, R³ isR^(3A)-substituted —O—C₅ alkyl. In embodiments, R³ is R^(3A)-substituted—O—C₆ alkyl. In embodiments, R³ is R^(3A)-substituted 2 to 10 memberedheteroalkyl. In embodiments, R³ is R^(3A)-substituted 2 to 8 memberedheteroalkyl. In embodiments, R³ is R^(3A)-substituted 2 to 6 memberedheteroalkyl. In embodiments, R³ is R^(3A)-substituted 2 to 4 memberedheteroalkyl. In embodiments, R³ is an unsubstituted 2 to 10 memberedheteroalkyl. In embodiments, R³ is an unsubstituted 2 to 8 memberedheteroalkyl. In embodiments, R³ is an unsubstituted 2 to 6 memberedheteroalkyl. In embodiments, R³ is an unsubstituted 2 to 4 memberedheteroalkyl.

R^(3A) is independently oxo, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CHCl₂,—CHBr₂, —CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I, —CN, —OH, —NH₂,—COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NH₄NH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃,—OCF₃, —OCBr₃, —OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl,—OCH₂Br, —OCH₂I, —OCH₂F, —N₃, —SF₅, —NH₃ ⁺, —SO₃ ⁻, —OPO₃H⁻, —SCN,—ONO₂, unsubstituted alkyl (e.g., C₁-C₂₀, C₁₀-C₂₀, C₁-C₈, C₁-C₆, orC₁-C₄), unsubstituted heteroalkyl (e.g., 2 to 20, 8 to 20, 2 to 10, 2 to8, 2 to 6, or 2 to 4 membered), unsubstituted cycloalkyl (e.g., C₃-C₈,C₃-C₆, or C₅-C₆), unsubstituted heterocycloalkyl (e.g., 3 to 8, 3 to 6,or 5 to 6 membered), unsubstituted aryl (e.g., C₆-C₁₀, C₁₀, or phenyl),or unsubstituted heteroaryl (e.g., 5 to 10, 5 to 9, or 5 to 6 membered).In embodiments, R^(3A) is —OH.

In embodiments, R⁴ is R^(4A)-substituted or unsubstituted alkyl (e.g.,C₁-C₂₀, C₁₀-C₂₀, C₁-C₈, C₁-C₆, or C₁-C₄), or R^(4A)-substituted orunsubstituted heteroalkyl (e.g., 2 to 20, 8 to 20, 2 to 10, 2 to 8, 2 to6, or 2 to 4 membered). In embodiments, R⁴ is substituted orunsubstituted alkyl (e.g., C₁-C₂₀, C₁₀-C₂₀, C₁-C₈, C₁-C₆, or C₁-C₄), orsubstituted or unsubstituted heteroalkyl (e.g., 2 to 20, 8 to 20, 2 to10, 2 to 8, 2 to 6, or 2 to 4 membered).

In embodiments, R⁴ is unsubstituted —O—C₁-C₆ or —O—C₁-C₄ alkyl. Inembodiments, R⁴ is unsubstituted —O—C₁-C₄ alkyl. In embodiments, R⁴ isunsubstituted —O—C₁-C₆ alkyl. In embodiments, R⁴ is unsubstituted—O-methyl. In embodiments, R⁴ is unsubstituted —O—C₂ alkyl. Inembodiments, R⁴ is unsubstituted —O—C₃ alkyl. In embodiments, R⁴ isunsubstituted —O—C₄ alkyl. In embodiments, R⁴ is unsubstituted —O—C₅alkyl. In embodiments, R⁴ is unsubstituted —O—C₆ alkyl. In embodiments,R⁴ is R^(4A)-substituted —O—C₁-C₆ or —O—C₁-C₄ alkyl. In embodiments, R⁴is R^(4A)-substituted —O—C₁-C₄ alkyl. In embodiments, R⁴ isR^(4A)-substituted —O—C₁-C₆ alkyl. In embodiments, R⁴ isR^(4A)-substituted —O-methyl. In embodiments, R⁴ is R^(4A)-substituted—O—C₂ alkyl. In embodiments, R⁴ is R^(4A)-substituted —O—C₃ alkyl. Inembodiments, R⁴ is R^(4A)-substituted —O—C₄ alkyl. In embodiments, R⁴ isR⁴-substituted —O—C₅ alkyl. In embodiments, R⁴ is R^(4A)-substituted—O—C₆ alkyl. In embodiments, R⁴ is R^(4A)-substituted 2 to 10 memberedheteroalkyl. In embodiments, R⁴ is R^(4A)-substituted 2 to 8 memberedheteroalkyl. In embodiments, R⁴ is R^(4A)-substituted 2 to 6 memberedheteroalkyl. In embodiments, R⁴ is R^(4A)-substituted 2 to 4 memberedheteroalkyl. In embodiments, R⁴ is an unsubstituted 2 to 10 memberedheteroalkyl. In embodiments, R⁴ is an unsubstituted 2 to 8 memberedheteroalkyl. In embodiments, R⁴ is an unsubstituted 2 to 6 memberedheteroalkyl. In embodiments, R⁴ is an unsubstituted 2 to 4 memberedheteroalkyl.

R^(4A) is independently oxo, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CHCl₂,—CHBr₂, —CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I, —CN, —OH, —NH₂,—COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NH₄NH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃,—OCF₃, —OCBr₃, —OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl,—OCH₂Br, —OCH₂I, —OCH₂F, —N₃, —SF₅, —NH₃ ⁺, —SO₃ ⁻, —OPO₃H⁻, —SCN,—ONO₂, unsubstituted alkyl (e.g., C₁-C₂₀, C₁₀-C₂₀, C₁-C₈, C₁-C₆, orC₁-C₄), unsubstituted heteroalkyl (e.g., 2 to 20, 8 to 20, 2 to 10, 2 to8, 2 to 6, or 2 to 4 membered), unsubstituted cycloalkyl (e.g., C₃-C₈,C₃-C₆, or C₅-C₆), unsubstituted heterocycloalkyl (e.g., 3 to 8, 3 to 6,or 5 to 6 membered), unsubstituted aryl (e.g., C₆-C₁₀, C₁₀, or phenyl),or unsubstituted heteroaryl (e.g., 5 to 10, 5 to 9, or 5 to 6 membered).In embodiments, R^(4A) is —OH.

In embodiments, L¹ is L^(1A)-substituted or unsubstituted alkylene(e.g., C₁-C₂₀, C₁₀-C₂₀, C₁-C₈, C₁-C₆, or C₁-C₄), or L^(1A)-substitutedor unsubstituted heteroalkylene (e.g., 2 to 20, 8 to 20, 2 to 10, 2 to8, 2 to 6, or 2 to 4 membered). In embodiments, L¹ is substituted orunsubstituted alkylene (e.g., C₁-C₂₀, C₁₀-C₂₀, C₁-C₈, C₁-C₆, or C₁-C₄),or substituted or unsubstituted heteroalkylene (e.g., 2 to 20, 8 to 20,2 to 10, 2 to 8, 2 to 6, or 2 to 4 membered).

In embodiments, L¹ is unsubstituted C₁-C₆ or C₁-C₄ alkylene. Inembodiments, L¹ is unsubstituted C₁-C₄ alkylene. In embodiments, L¹ isunsubstituted C₁-C₆ alkylene. In embodiments, L¹ is unsubstitutedmethylene. In embodiments, L¹ is unsubstituted C₂ alkylene. Inembodiments, L¹ is unsubstituted C₃ alkylene. In embodiments, L¹ isunsubstituted C₄ alkylene. In embodiments, L¹ is unsubstituted C₅alkylene. In embodiments, L¹ is unsubstituted C₆ alkylene. Inembodiments, L¹ is L^(1A)-substituted C₁-C₆ or C₁-C₄ alkylene. Inembodiments, L¹ is L^(1A)-substituted C₁-C₄ alkylene. In embodiments, L¹is L^(1A)-substituted C₁-C₆ alkylene. In embodiments, L¹ isL^(1A)-substituted methylene. In embodiments, L¹ is L^(1A)-substitutedC₂ alkylene. In embodiments, L¹ is L^(1A)-substituted C₃ alkylene. Inembodiments, L¹ is L^(1A)-substituted C₄ alkylene. In embodiments, L¹ isL^(1A)-substituted C₅ alkylene. In embodiments, L¹ is L^(1A)-substitutedC₆ alkylene. In embodiments, L¹ is L^(1A)-substituted 2 to 10 memberedheteroalkylene. In embodiments, L¹ is L^(1A)-substituted 2 to 8 memberedheteroalkylene. In embodiments, L¹ is L^(1A)-substituted 2 to 6 memberedheteroalkylene. In embodiments, L¹ is L^(1A)-substituted 2 to 4 memberedheteroalkylene. In embodiments, L¹ is an unsubstituted 2 to 10 memberedheteroalkylene. In embodiments, L¹ is an unsubstituted 2 to 8 memberedheteroalkylene. In embodiments, L¹ is an unsubstituted 2 to 6 memberedheteroalkylene. In embodiments, L¹ is an unsubstituted 2 to 4 memberedheteroalkylene.

L^(1A) is independently oxo, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CHCl₂,—CHBr₂, —CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I, —CN, —OH, —NH₂,—COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NH—NH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃,—OCF₃, —OCBr₃, —OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl,—OCH₂Br, —OCH₂I, —OCH₂F, —N₃, —SF₅, —NH₃ ⁺, —SO₃ ⁻, —OPO₃H⁻, —SCN,—ONO₂, unsubstituted alkylene (e.g., C₁-C₂₀, C₁₀-C₂₀, C₁-C₈, C₁-C₆, orC₁-C₄), unsubstituted heteroalkylene (e.g., 2 to 20, 8 to 20, 2 to 10, 2to 8, 2 to 6, or 2 to 4 membered), unsubstituted cycloalkylene (e.g.,C₃-C₈, C₃-C₆, or C₅-C₆), unsubstituted heterocycloalkylene (e.g., 3 to8, 3 to 6, or 5 to 6 membered), unsubstituted arylene (e.g., C₆-C₁₀,C₁₀, or phenylene), or unsubstituted heteroarylene (e.g., 5 to 10, 5 to9, or 5 to 6 membered). In embodiments, L^(1A) is —CH₃.

In embodiments, L^(1A) is independently oxo, halogen, —CCl₃, —CBr₃,—CF₃, —CI₃, —CHCl₂, —CHBr₂, —CHF₂, —CHI₂, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I,—CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂,—ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH,—OCCl₃, —OCF₃, —OCBr₃, —OCI₃, —OCHCl₂, —OCHBr₂, —OCHI₂, —OCHF₂, —OCH₂Cl,—OCH₂Br, —OCH₂I, —OCH₂F, —N₃, —SF₅, —NH₃ ⁺, —SO₃ ⁻, —OPO₃H⁻, —SCN,—ONO₂, unsubstituted alkyl (e.g., C₁-C₂₀, C₁₀-C₂₀, C₁-C₈, C₁-C₆, orC₁-C₄), unsubstituted heteroalkyl (e.g., 2 to 20, 8 to 20, 2 to 10, 2 to8, 2 to 6, or 2 to 4 membered), unsubstituted cycloalkyl (e.g., C₃-C₈,C₃-C₆, or C₅-C₆), unsubstituted heterocycloalkyl (e.g., 3 to 8, 3 to 6,or 5 to 6 membered), unsubstituted aryl (e.g., C₆-C₁₀, C₁₀, or phenyl),or unsubstituted heteroaryl (e.g., 5 to 10, 5 to 9, or 5 to 6 membered).In embodiments, L^(1A) is —CH₃.

In embodiments, the polymer coating includes polymerized units of3-(trimethoxysilyl)propyl methacrylate (TMSPM)

(trimethoxysilyl)propyl methacrylate (TMSPA)

3-(triethoxysilyl)propyl methacrylate (TESPM)

3-(triethoxysilyl)propyl acrylate (TESPA)

3-(dimethoxy(1-methylethoxy)silyl]propyl methacrylate

3-(ethoxydimethoxysilyl)propyl 2-methyl-2-propenoate

3-(Tripropoxysilyl)propyl 2-methyl-2-propenoate

2-Methyl-3-(triethoxysilyl)propyl 2-methyl-2-propenoate

3 (Methyldipropoxysilyl)propyl 2-methyl-2-propenoate

3-(Diethoxymethylsilyl)propyl 2-methyl-2-propenoate

3-[Diethoxy(2-hydroxyethoxy)silyl]propyl 2-methyl-2-propenoate

or 3-(Butyldimethoxysilyl)propyl 2-methyl-2-propenoate

In embodiments, the polymer coating includes polymerized units ofglycidyloxypropyl trimethoxysilane (GPTMS). In embodiments, the polymercoating includes polymerized units of alkoxysilyl polymers andpolymerized units of polyethylene glycol methacrylate (PEGMA).

In embodiments, the average longest dimension of the nanoparticle isfrom about 100 nm to about 400 nm. In embodiments, the average longestdimension of the nanoparticle is about 75 nm, 80 nm, 85 nm, 90 nm, 95nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140nm, 145 nm, 150 nm, 155 nm, 160 nm, 165 nm, 170 nm, 175 nm, 180 nm, 185nm, 190 nm, 195 nm, 200 nm, 205 nm, 210 nm, 215 nm, 220 nm, 225 nm, 230nm, 235 nm, 240 nm, 245 nm, 250 nm, 255 nm, 260 nm, 265 nm, 270 nm, 275nm, 280 nm, 285 nm, 290 nm, 295 nm, 300 nm, 305 nm, 310 nm, 315 nm, 320nm, 325 nm, 330 nm, 335 nm, 340 nm, 345 nm, 350 nm, 355 nm, 360 nm, 365nm, 370 nm, 375 nm, 380 nm, 385 nm, 390 nm, 395 nm, 400 nm, 405 nm, 410nm, 415 nm, 420 nm, 425 nm, 430 nm, 435 nm, 440 nm, 445 nm, 450 nm, 455nm, 460 nm, 465 nm, 470 nm, 475 nm, 480 nm, 485 nm, 490 nm, 495 nm, 500nm, 505 nm, 510 nm, 515 nm, 520 nm, 525 nm, 530 nm, 535 nm, 540 nm, 545nm, 550 nm, 555 nm, 560 nm, 565 nm, 570 nm, 575 nm, 580 nm, 585 nm, 590nm, 595 nm, or 600 nm. In embodiments, the average longest dimension ofthe nanoparticle is from about 600 nm, 605 nm, 610 nm, 615 nm, 620 nm,625 nm, 630 nm, 635 nm, 640 nm, 645 nm, 650 nm, 655 nm, 660 nm, 665 nm,670 nm, 675 nm, 680 nm, 685 nm, 690 nm, 695 nm, 700 nm, 705 nm, 710 nm,715 nm, 720 nm, 725 nm, 730 nm, 735 nm, 740 nm, 745 nm, 750 nm, 755 nm,760 nm, 765 nm, 770 nm, 775 nm, 780 nm, 785 nm, 790 nm, 795 nm, 800 nm,805 nm, 810 nm, 815 nm, 820 nm, 825 nm, 830 nm, 835 nm, 840 nm, 845 nm,850 nm, 855 nm, 860 nm, 865 nm, 870 nm, 875 nm, 880 nm, 885 nm, 890 nm,895 nm, 900 nm, 905 nm, 910 nm, 915 nm, 920 nm, 925 nm, 930 nm, 935 nm,940 nm, 945 nm, 950 nm, 955 nm, 960 nm, 965 nm, 970 nm, 975 nm, 980 nm,985 nm, 990 nm, 995 nm or about 1000 nm. In embodiments, the averagelongest dimension of the nanoparticle is less than about 1000 nm. Inembodiments, the average longest dimension of the nanoparticle is lessthan about 900 nm. In embodiments, the average longest dimension of thenanoparticle is less than about 800 nm. In embodiments, the averagelongest dimension of the nanoparticle is less than about 700 nm. Inembodiments, the average longest dimension of the nanoparticle is lessthan about 600 nm. In embodiments, the average longest dimension of thenanoparticle is less than about 500 nm. In embodiments, the averagelongest dimension of the nanoparticle is less than about 400 nm. Inembodiments, the average longest dimension of the nanoparticle is lessthan about 300 nm. In embodiments, the average longest dimension of thenanoparticle is less than about 200 nm. In embodiments, the averagelongest dimension of the nanoparticle is less than about 100 nm. Inembodiments, the average longest dimension of the nanoparticle is 400 nmwithout the particle shell. In embodiments, the average longestdimension of the nanoparticle is about 550 to about 650 nm with theparticle shell. In embodiments, the average longest dimension of thenanoparticle is about 580 to about 650 nm with the particle shellcontaining immobilized oligonucleotides.

In some embodiments, the average longest dimension of the particle isfrom about 200 nm to about 1000 nm. In embodiments, the average longestdimension of the particle is from about 150 nm to about 600 nm. In someembodiments, the average longest dimension of the particle is from about350 nm to about 600 nm. In some embodiments, the average longestdimension of the particle is from about 400 nm to about 500 nm. In someembodiments, the average longest dimension of the particle is about 500nm. In some embodiments, the average longest dimension of the particleis about 400 nm. In some embodiments, the average longest dimension ofthe particle is about 400 nm, 450 nm, 500 nm, or 550 nm. In someembodiments, the average longest dimension of the particle is about 410nm, 420 nm, 430 nm, 440 nm or 450 nm. In some embodiments, the averagelongest dimension of the particle is about 460 nm, 470 nm, 480 nm, 490nm or 500 nm. In embodiments, the average longest dimension of theparticle is at least, about, or at most 100, 200, 300, 400, 500, 600,700, 800, 900, 1000 nm, or a number or a range between any two of thesevalues. In embodiments, the shell diameter is about 0.1-10 microns,0.25-5 microns, 0.5-2 microns, 1 micron, or a number or a range betweenany two of these values. In embodiments, the particle shell diameter isat least, about, or at most 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9,1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3,2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7,3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0 μm or anumber or a range between any two of these values. In embodiments, thecore diameter is about 150-700 nanometers, and/or the shell diameter(alternatively referred to as the particle polymer; see for example FIG.10A) is about 0.25-5 μm (microns).

In some embodiments, the wells of the array are separated from eachother by about 0.2 μm to about 2.0 μm. In some embodiments, the wells ofthe array are separated from each other by about 0.7 μm to about 1.5 μm.In some embodiments, the wells of the array are separated from eachother by at least or at most 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9,1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 μm. In someembodiments, the wells of the array are from about 0.2 μm to about 2 μmin diameter, and wherein the wells of the array are about 0.5 μm toabout 2 μm in depth. In some embodiments, the wells of the array arefrom about 0.2 μm to about 2 μm in diameter, and wherein the wells ofthe array are about 0.5 μm to about 1.5 μm in depth. Each well of themultiwell container is capable of retaining a volume of liquid. Forexample, the volume of the wells can be at least about 1×10⁻³ μm³, about1×10⁻² μm³, about 0.1 μm³, about 1 μm³, about 10 μm³, about 100 μm³, ormore. In embodiments, the volume of the wells can be at most about 1×10⁴μm³, about 1×10³ μm³, about 100 μm³, about 10 μm³, about 1 μm³, about0.1 μm³, or less. In embodiments, the depth of the well is measured fromthe bottom of the well to the top of the array. In embodiments, thedepth of the well is measured from the bottom of the well to the top ofthe interstitial region. In embodiments, the depth of the well ismeasured from the bottom of the well to the top of the photoresist. Inembodiments, the array is a nanoarray which can have nanowells having adiameter sufficient to allow only one particle into the well. It isunderstood that the size of the nanowell will be dependent upon the sizeof the particle. In some embodiments, the diameter of the nanowells areless than 700 nm, less than 600 nm, less than 500 nm, less than 400 nm,less than 300 nm, less than 200 nm, or less than 100 nm. It is alsounderstood that the size of the wells on the array can be of varioussizes and will ultimately depend on the systems and/or apparatus used toanalyze later reactions.

In some embodiments, greater than 50%, 60%, 70%, 80%, 90% or 95% of thewells include a particle. In some embodiments, greater than 50% of thewells include a particle. In some embodiments, greater than 60% of thewells include a particle. In some embodiments, greater than 70% of thewells include a particle. In some embodiments, greater than 80% of thewells include a particle. In some embodiments, greater than 90% of thewells include a particle. In some embodiments, greater than 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the wells include aparticle. In some embodiments, about 50%, 60%, 70%, 80%, 90% or 95% ofthe wells include a particle. In some embodiments, about 50% of thewells include a particle. In some embodiments, about 60% of the wellsinclude a particle. In some embodiments, about 70% of the wells includea particle. In some embodiments, about 80% of the wells include aparticle. In some embodiments, about 90% of the wells include aparticle. In some embodiments, about 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% of the wells include a particle. In embodiments, one ormore wells has two or more particles in each well. In embodiments, eachwell has two or more particles per well. In embodiments, one or morewells has only one particle in each well. In embodiments, each well has0 to 1 particles per well. In embodiments, each well has 0 to 3particles per well.

In some embodiments, the interstitial regions are substantially free ofoligonucleotide moieties. In some embodiments, the interstitial regionsare substantially free of particles. In embodiments, the interstitialregions are substantially free of polynucleotides. In embodiments, theinterstitial regions are substantially free of a polymer. Inembodiments, the interstitial regions include a photoresist. Inembodiments, physical removal (e.g., wiping the surface of the solidsupport) of any excess polymer and particles ensures the interstitialregions are substantially free of oligonucleotide moieties and/orparticles. In embodiments, substantially free includes a trace amount oran undetectable amount.

In embodiments, each particle includes a plurality of oligonucleotidemoieties covalently attached to said particle via a polymericbioconjugate linker. In embodiments, the polymeric bioconjugate linkeris a polymer (i.e., a molecule including structurally unique repeatingunits) including one or more reacted bioconjugate reactive moieties. Inembodiments, the bioconjugate linker is illustrated in Scheme 1. Inembodiments, the polymeric bioconjugate linker is a polymer including asubunit of formula Ia, Ib, II, or III as described in U.S. Pat. No.11,236,387, which is incorporated herein by reference in its entirety.

In embodiments, the polymeric bioconjugate linker is polymer including asubunit having the formula:

R²² is independently an oligonucleotide moiety. R²¹, R²³, R⁵, R⁶, R⁷,and R⁸ are independently hydrogen, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃,—CH₂Cl, —CH₂Br, —CH₂F, —CH₂I, —CHCl₂, —CHBr₂, —CHF₂, —CHI₂, —CN, —OH,—NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃,—OCBr₃, —OCF₃, —OCI₃, —OCH₂Cl, —OCH₂Br, —OCH₂F, —OCH₂I, —OCHCl₂,—OCHBr₂, —OCHF₂, —OCHI₂, substituted or unsubstituted alkyl (e.g.,C₁-C₈, C₁-C₆, C₁-C₄, or C₁-C₂), substituted or unsubstituted heteroalkyl(e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3membered, or 4 to 5 membered), substituted or unsubstituted cycloalkyl(e.g., C₃-C₈, C₃-C₆, C₄-C₆, or C₅-C₆), substituted or unsubstitutedheterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6membered, 4 to 5 membered, or 5 to 6 membered), substituted orunsubstituted aryl (e.g., C₆-C₁₀ or phenyl), or substituted orunsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to 9 membered, or 5to 6 membered).

L² is independently -L^(2A)-L^(2B)-L^(2C)-L^(2D)-L^(2E)- or abioconjugate linker. L^(2A), L^(2B) L^(2C), L^(2D), and L^(2E) areindependently a bond, a bioconjugate linker, —S(O)₂—, —S(O)—, —S(O)₂NH—,—NH—, —O—, —S—, —SS—, —C(O)—, —C(O)NH—, —C(O)CH₂—, —NHC(O)—, —NHC(O)NH—,—C(O)O—, —OC(O)—, substituted or unsubstituted alkylene (e.g., C₁-C₈,C₁-C₆, C₁-C₄, or C₁-C₂), substituted or unsubstituted heteroalkylene(e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3membered, or 4 to 5 membered), substituted or unsubstitutedcycloalkylene (e.g., C₃-C₈, C₃-C₆, C₄-C₆, or C₅-C₆), substituted orunsubstituted heterocycloalkylene (e.g., 3 to 8 membered, 3 to 6membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered),substituted or unsubstituted arylene (e.g., C₆-C₁₀ or phenylene), orsubstituted or unsubstituted heteroarylene (e.g., 5 to 10 membered, 5 to9 membered, or 5 to 6 membered). In embodiments, L² is a bioconjugatelinker.

L⁴ is independently -L^(4A)-L^(4B)-L^(4C)-L^(4D)-L^(4E)-. L^(4A), L^(4B)L^(4C), L^(4D), and L^(4E) are independently a bond, —S(O)₂—, —S(O)—,—S(O)₂NH—, —NH—, —O—, —S—, —C(O)—, —C(O)NH—, —C(O)CH₂—, —NHC(O)—,—NHC(O)NH—, —C(O)O—, —OC(O)—, substituted or unsubstituted alkylene(e.g., C₁-C₈, C₁-C₆, C₁-C₄, or C₁-C₂), substituted or unsubstitutedheteroalkylene (e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6 membered,2 to 3 membered, or 4 to 5 membered), substituted or unsubstitutedcycloalkylene (e.g., C₃-C₈, C₃-C₆, C₄-C₆, or C₅-C₆), substituted orunsubstituted heterocycloalkylene (e.g., 3 to 8 membered, 3 to 6membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered),substituted or unsubstituted arylene (e.g., C₆-C₁₀ or phenylene), orsubstituted or unsubstituted heteroarylene (e.g., 5 to 10 membered, 5 to9 membered, or 5 to 6 membered).

R²⁴ is independently hydrogen, halogen, —CX²⁴ ₃, —CHX²⁴ ₂, —CH₂X²⁴,—OCX²⁴ ₃, —OCH₂X²⁴, —OCHX²⁴ ₂, —CN, —SO_(n24)R^(24D),—SO_(v24)NR^(24A)R^(24B), NHC(O)NR^(24A)R^(24B), —N(O)_(m24),—NR^(24A)R^(24B), —C(O)R^(24C), —C(O)—OR^(24C), —C(O)NR^(24A)R^(24B),—OR^(24D), —NR^(24A)SO₂R^(24D), —NR^(24A)C(O)R^(24C),—NR^(24A)C(O)OR^(24C), —NR^(24A)OR^(24C), —OC(O)R^(24C), substituted orunsubstituted alkyl (e.g., C₁-C₈, C₁-C₆, C₁-C₄, or C₁-C₂), substitutedor unsubstituted heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered, 4to 6 membered, 2 to 3 membered, or 4 to 5 membered), substituted orunsubstituted cycloalkyl (e.g., C₃-C₈, C₃-C₆, C₄-C₆, or C₅-C₆),substituted or unsubstituted heterocycloalkyl (e.g., 3 to 8 membered, 3to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered),substituted or unsubstituted aryl (e.g., C₆-C₁₀ or phenyl), orsubstituted or unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to 9membered, or 5 to 6 membered); wherein R²⁴ is a first non-reactivemoiety.

R^(24A), R^(24B), R^(24C), and R^(24D) are independently hydrogen, —CX₃,—CN, —COOH, —CONH₂, —CHX₂, —CH₂X, substituted or unsubstituted alkyl(e.g., C₁-C₈, C₁-C₆, C₁-C₄, or C₁-C₂), substituted or unsubstitutedheteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2to 3 membered, or 4 to 5 membered), substituted or unsubstitutedcycloalkyl (e.g., C₃-C₈, C₃-C₆, C₄-C₆, or C₅-C₆), substituted orunsubstituted heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered,4 to 6 membered, 4 to 5 membered, or 5 to 6 membered), substituted orunsubstituted aryl (e.g., C₆-C₁₀ or phenyl), substituted orunsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to 9 membered, or 5to 6 membered), a protecting group, or a leaving group; R^(24A) andR^(24B) substituents bonded to the same nitrogen atom may optionally bejoined to form a substituted or unsubstituted heterocycloalkyl (e.g., 3to 8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5to 6 membered) or substituted or unsubstituted heteroaryl (e.g., 5 to 10membered, 5 to 9 membered, or 5 to 6 membered).

L⁹ is independently -L^(9A)-L^(9B)-L^(9C)-L^(9D)-L^(9E)-. L^(9A), L^(9B)L^(9C), L^(9D), and L^(9E) are independently a bond, —S(O)₂—, —S(O)—,—S(O)₂NH—, —NH—, —O—, —S—, —SS—, —C(O)—, —C(O)NH—, —C(O)CH₂—, —NHC(O)—,—NHC(O)NH—, —C(O)O—, —OC(O)—, substituted or unsubstituted alkylene(e.g., C₁-C₈, C₁-C₆, C₁-C₄, or C₁-C₂), substituted or unsubstitutedheteroalkylene (e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6 membered,2 to 3 membered, or 4 to 5 membered), substituted or unsubstitutedcycloalkylene (e.g., C₃-C₈, C₃-C₆, C₄-C₆, or C₅-C₆), substituted orunsubstituted heterocycloalkylene (e.g., 3 to 8 membered, 3 to 6membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered),substituted or unsubstituted arylene (e.g., C₆-C₁₀ or phenylene), orsubstituted or unsubstituted heteroarylene (e.g., 5 to 10 membered, 5 to9 membered, or 5 to 6 membered).

R⁹ is independently an oligonucleotide or a second non-reactive moiety.The symbol z2 is independently an integer from 1 to 5000. The symbolsz1a, z1b, z1c, and z1d are each independently an integer from 0 to 5000.X and X²⁴ are independently —F, —Cl, —Br, or —I. The symbol n24 isindependently an integer from 0 to 4. The symbols m24 and v24 are eachindependently an integer from 1 to 2.

In embodiments, a substituted R²¹ (e.g., substituted alkyl, substitutedheteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl,substituted aryl, and/or substituted heteroaryl) is substituted with atleast one substituent group, size-limited substituent group, or lowersubstituent group; wherein if the substituted R²¹ is substituted with aplurality of groups selected from substituent groups, size-limitedsubstituent groups, and lower substituent groups; each substituentgroup, size-limited substituent group, and/or lower substituent groupmay optionally be different. In embodiments, when R²¹ is substituted, itis substituted with at least one substituent group. In embodiments, whenR²¹ is substituted, it is substituted with at least one size-limitedsubstituent group. In embodiments, when R²¹ is substituted, it issubstituted with at least one lower substituent group.

In embodiments, a substituted R²³ (e.g., substituted alkyl, substitutedheteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl,substituted aryl, and/or substituted heteroaryl) is substituted with atleast one substituent group, size-limited substituent group, or lowersubstituent group; wherein if the substituted R²³ is substituted with aplurality of groups selected from substituent groups, size-limitedsubstituent groups, and lower substituent groups; each substituentgroup, size-limited substituent group, and/or lower substituent groupmay optionally be different. In embodiments, when R²³ is substituted, itis substituted with at least one substituent group. In embodiments, whenR²³ is substituted, it is substituted with at least one size-limitedsubstituent group. In embodiments, when R²³ is substituted, it issubstituted with at least one lower substituent group.

In embodiments, a substituted R⁵ (e.g., substituted alkyl, substitutedheteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl,substituted aryl, and/or substituted heteroaryl) is substituted with atleast one substituent group, size-limited substituent group, or lowersubstituent group; wherein if the substituted R⁵ is substituted with aplurality of groups selected from substituent groups, size-limitedsubstituent groups, and lower substituent groups; each substituentgroup, size-limited substituent group, and/or lower substituent groupmay optionally be different. In embodiments, when R⁵ is substituted, itis substituted with at least one substituent group. In embodiments, whenR⁵ is substituted, it is substituted with at least one size-limitedsubstituent group. In embodiments, when R⁵ is substituted, it issubstituted with at least one lower substituent group.

In embodiments, a substituted R⁶ (e.g., substituted alkyl, substitutedheteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl,substituted aryl, and/or substituted heteroaryl) is substituted with atleast one substituent group, size-limited substituent group, or lowersubstituent group; wherein if the substituted R⁶ is substituted with aplurality of groups selected from substituent groups, size-limitedsubstituent groups, and lower substituent groups; each substituentgroup, size-limited substituent group, and/or lower substituent groupmay optionally be different. In embodiments, when R⁶ is substituted, itis substituted with at least one substituent group. In embodiments, whenR⁶ is substituted, it is substituted with at least one size-limitedsubstituent group. In embodiments, when R⁶ is substituted, it issubstituted with at least one lower substituent group.

In embodiments, a substituted R⁷ (e.g., substituted alkyl, substitutedheteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl,substituted aryl, and/or substituted heteroaryl) is substituted with atleast one substituent group, size-limited substituent group, or lowersubstituent group; wherein if the substituted R⁷ is substituted with aplurality of groups selected from substituent groups, size-limitedsubstituent groups, and lower substituent groups; each substituentgroup, size-limited substituent group, and/or lower substituent groupmay optionally be different. In embodiments, when R⁷ is substituted, itis substituted with at least one substituent group. In embodiments, whenR⁷ is substituted, it is substituted with at least one size-limitedsubstituent group. In embodiments, when R⁷ is substituted, it issubstituted with at least one lower substituent group.

In embodiments, a substituted R⁸ (e.g., substituted alkyl, substitutedheteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl,substituted aryl, and/or substituted heteroaryl) is substituted with atleast one substituent group, size-limited substituent group, or lowersubstituent group; wherein if the substituted R⁸ is substituted with aplurality of groups selected from substituent groups, size-limitedsubstituent groups, and lower substituent groups; each substituentgroup, size-limited substituent group, and/or lower substituent groupmay optionally be different. In embodiments, when R⁸ is substituted, itis substituted with at least one substituent group. In embodiments, whenR⁸ is substituted, it is substituted with at least one size-limitedsubstituent group. In embodiments, when R⁸ is substituted, it issubstituted with at least one lower substituent group.

In embodiments, a substituted L^(2A) (e.g., substituted alkylene,substituted heteroalkylene, substituted cycloalkylene, substitutedheterocycloalkylene, substituted arylene, and/or substitutedheteroarylene) is substituted with at least one substituent group,size-limited substituent group, or lower substituent group; wherein ifthe substituted L^(2A) is substituted with a plurality of groupsselected from substituent groups, size-limited substituent groups, andlower substituent groups; each substituent group, size-limitedsubstituent group, and/or lower substituent group may optionally bedifferent. In embodiments, when L^(2A) is substituted, it is substitutedwith at least one substituent group. In embodiments, when L^(2A) issubstituted, it is substituted with at least one size-limitedsubstituent group. In embodiments, when L^(2A) is substituted, it issubstituted with at least one lower substituent group.

In embodiments, a substituted L^(2B) (e.g., substituted alkylene,substituted heteroalkylene, substituted cycloalkylene, substitutedheterocycloalkylene, substituted arylene, and/or substitutedheteroarylene) is substituted with at least one substituent group,size-limited substituent group, or lower substituent group; wherein ifthe substituted L^(2B) is substituted with a plurality of groupsselected from substituent groups, size-limited substituent groups, andlower substituent groups; each substituent group, size-limitedsubstituent group, and/or lower substituent group may optionally bedifferent. In embodiments, when L^(2B) is substituted, it is substitutedwith at least one substituent group. In embodiments, when L^(2B) issubstituted, it is substituted with at least one size-limitedsubstituent group. In embodiments, when L^(2B) is substituted, it issubstituted with at least one lower substituent group.

In embodiments, a substituted L^(2C) (e.g., substituted alkylene,substituted heteroalkylene, substituted cycloalkylene, substitutedheterocycloalkylene, substituted arylene, and/or substitutedheteroarylene) is substituted with at least one substituent group,size-limited substituent group, or lower substituent group; wherein ifthe substituted L^(2C) is substituted with a plurality of groupsselected from substituent groups, size-limited substituent groups, andlower substituent groups; each substituent group, size-limitedsubstituent group, and/or lower substituent group may optionally bedifferent. In embodiments, when L^(2C) is substituted, it is substitutedwith at least one substituent group. In embodiments, when L^(2C) issubstituted, it is substituted with at least one size-limitedsubstituent group. In embodiments, when L^(2C) is substituted, it issubstituted with at least one lower substituent group.

In embodiments, a substituted L^(2D) (e.g., substituted alkylene,substituted heteroalkylene, substituted cycloalkylene, substitutedheterocycloalkylene, substituted arylene, and/or substitutedheteroarylene) is substituted with at least one substituent group,size-limited substituent group, or lower substituent group; wherein ifthe substituted L^(2D) is substituted with a plurality of groupsselected from substituent groups, size-limited substituent groups, andlower substituent groups; each substituent group, size-limitedsubstituent group, and/or lower substituent group may optionally bedifferent. In embodiments, when L^(2D) is substituted, it is substitutedwith at least one substituent group. In embodiments, when L^(2D) issubstituted, it is substituted with at least one size-limitedsubstituent group. In embodiments, when L^(2D) is substituted, it issubstituted with at least one lower substituent group.

In embodiments, a substituted L^(2E) (e.g., substituted alkylene,substituted heteroalkylene, substituted cycloalkylene, substitutedheterocycloalkylene, substituted arylene, and/or substitutedheteroarylene) is substituted with at least one substituent group,size-limited substituent group, or lower substituent group; wherein ifthe substituted L^(2E) is substituted with a plurality of groupsselected from substituent groups, size-limited substituent groups, andlower substituent groups; each substituent group, size-limitedsubstituent group, and/or lower substituent group may optionally bedifferent. In embodiments, when L^(2E) is substituted, it is substitutedwith at least one substituent group. In embodiments, when L^(2E) issubstituted, it is substituted with at least one size-limitedsubstituent group. In embodiments, when L^(2E) is substituted, it issubstituted with at least one lower substituent group.

In embodiments, a substituted L^(4A) (e.g., substituted alkylene,substituted heteroalkylene, substituted cycloalkylene, substitutedheterocycloalkylene, substituted arylene, and/or substitutedheteroarylene) is substituted with at least one substituent group,size-limited substituent group, or lower substituent group; wherein ifthe substituted L^(4A) is substituted with a plurality of groupsselected from substituent groups, size-limited substituent groups, andlower substituent groups; each substituent group, size-limitedsubstituent group, and/or lower substituent group may optionally bedifferent. In embodiments, when L^(4A) is substituted, it is substitutedwith at least one substituent group. In embodiments, when L^(4A) issubstituted, it is substituted with at least one size-limitedsubstituent group. In embodiments, when L^(4A) is substituted, it issubstituted with at least one lower substituent group.

In embodiments, a substituted L^(4B) (e.g., substituted alkylene,substituted heteroalkylene, substituted cycloalkylene, substitutedheterocycloalkylene, substituted arylene, and/or substitutedheteroarylene) is substituted with at least one substituent group,size-limited substituent group, or lower substituent group; wherein ifthe substituted L^(4B) is substituted with a plurality of groupsselected from substituent groups, size-limited substituent groups, andlower substituent groups; each substituent group, size-limitedsubstituent group, and/or lower substituent group may optionally bedifferent. In embodiments, when L^(4B) is substituted, it is substitutedwith at least one substituent group. In embodiments, when L^(4B) issubstituted, it is substituted with at least one size-limitedsubstituent group. In embodiments, when L^(4B) is substituted, it issubstituted with at least one lower substituent group.

In embodiments, a substituted L⁴c (e.g., substituted alkylene,substituted heteroalkylene, substituted cycloalkylene, substitutedheterocycloalkylene, substituted arylene, and/or substitutedheteroarylene) is substituted with at least one substituent group,size-limited substituent group, or lower substituent group; wherein ifthe substituted L^(4C) is substituted with a plurality of groupsselected from substituent groups, size-limited substituent groups, andlower substituent groups; each substituent group, size-limitedsubstituent group, and/or lower substituent group may optionally bedifferent. In embodiments, when L^(4C) is substituted, it is substitutedwith at least one substituent group. In embodiments, when L^(4C) issubstituted, it is substituted with at least one size-limitedsubstituent group. In embodiments, when L^(4C) is substituted, it issubstituted with at least one lower substituent group.

In embodiments, a substituted L^(4D) (e.g., substituted alkylene,substituted heteroalkylene, substituted cycloalkylene, substitutedheterocycloalkylene, substituted arylene, and/or substitutedheteroarylene) is substituted with at least one substituent group,size-limited substituent group, or lower substituent group; wherein ifthe substituted L^(4D) is substituted with a plurality of groupsselected from substituent groups, size-limited substituent groups, andlower substituent groups; each substituent group, size-limitedsubstituent group, and/or lower substituent group may optionally bedifferent. In embodiments, when L^(4D) is substituted, it is substitutedwith at least one substituent group. In embodiments, when L^(4D) issubstituted, it is substituted with at least one size-limitedsubstituent group. In embodiments, when L^(4D) is substituted, it issubstituted with at least one lower substituent group.

In embodiments, a substituted L^(4E) (e.g., substituted alkylene,substituted heteroalkylene, substituted cycloalkylene, substitutedheterocycloalkylene, substituted arylene, and/or substitutedheteroarylene) is substituted with at least one substituent group,size-limited substituent group, or lower substituent group; wherein ifthe substituted L^(4E) is substituted with a plurality of groupsselected from substituent groups, size-limited substituent groups, andlower substituent groups; each substituent group, size-limitedsubstituent group, and/or lower substituent group may optionally bedifferent. In embodiments, when L^(4E) is substituted, it is substitutedwith at least one substituent group. In embodiments, when L^(4E) issubstituted, it is substituted with at least one size-limitedsubstituent group. In embodiments, when L^(4E) is substituted, it issubstituted with at least one lower substituent group.

In embodiments, a substituted R²⁴ (e.g., substituted alkyl, substitutedheteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl,substituted aryl, and/or substituted heteroaryl) is substituted with atleast one substituent group, size-limited substituent group, or lowersubstituent group; wherein if the substituted R²⁴ is substituted with aplurality of groups selected from substituent groups, size-limitedsubstituent groups, and lower substituent groups; each substituentgroup, size-limited substituent group, and/or lower substituent groupmay optionally be different. In embodiments, when R²⁴ is substituted, itis substituted with at least one substituent group. In embodiments, whenR²⁴ is substituted, it is substituted with at least one size-limitedsubstituent group. In embodiments, when R²⁴ is substituted, it issubstituted with at least one lower substituent group.

In embodiments, a substituted R^(24A) (e.g., substituted alkyl,substituted heteroalkyl, substituted cycloalkyl, substitutedheterocycloalkyl, substituted aryl, and/or substituted heteroaryl) issubstituted with at least one substituent group, size-limitedsubstituent group, or lower substituent group; wherein if thesubstituted R^(24A) is substituted with a plurality of groups selectedfrom substituent groups, size-limited substituent groups, and lowersubstituent groups; each substituent group, size-limited substituentgroup, and/or lower substituent group may optionally be different. Inembodiments, when R^(24A) is substituted, it is substituted with atleast one substituent group. In embodiments, when R^(24A) issubstituted, it is substituted with at least one size-limitedsubstituent group. In embodiments, when R^(24A) is substituted, it issubstituted with at least one lower substituent group.

In embodiments, a substituted R^(24B) (e.g., substituted alkyl,substituted heteroalkyl, substituted cycloalkyl, substitutedheterocycloalkyl, substituted aryl, and/or substituted heteroaryl) issubstituted with at least one substituent group, size-limitedsubstituent group, or lower substituent group; wherein if thesubstituted R^(24B) is substituted with a plurality of groups selectedfrom substituent groups, size-limited substituent groups, and lowersubstituent groups; each substituent group, size-limited substituentgroup, and/or lower substituent group may optionally be different. Inembodiments, when R^(24B) is substituted, it is substituted with atleast one substituent group. In embodiments, when R^(24B) issubstituted, it is substituted with at least one size-limitedsubstituent group. In embodiments, when R^(24B) is substituted, it issubstituted with at least one lower substituent group.

In embodiments, a substituted ring formed when R^(24A) and R^(24B)substituents bonded to the same nitrogen atom are joined (e.g.,substituted heterocycloalkyl and/or substituted heteroaryl) issubstituted with at least one substituent group, size-limitedsubstituent group, or lower substituent group; wherein if thesubstituted ring formed when R^(24A) and R^(24B) substituents bonded tothe same nitrogen atom are joined is substituted with a plurality ofgroups selected from substituent groups, size-limited substituentgroups, and lower substituent groups; each substituent group,size-limited substituent group, and/or lower substituent group mayoptionally be different. In embodiments, when the substituted ringformed when R^(24A) and R^(24B) substituents bonded to the same nitrogenatom are joined is substituted, it is substituted with at least onesubstituent group. In embodiments, when the substituted ring formed whenR^(24A) and R^(24B) substituents bonded to the same nitrogen atom arejoined is substituted, it is substituted with at least one size-limitedsubstituent group. In embodiments, when the substituted ring formed whenR^(24A) and R^(24B) substituents bonded to the same nitrogen atom arejoined is substituted, it is substituted with at least one lowersubstituent group.

In embodiments, a substituted R^(24C) (e.g., substituted alkyl,substituted heteroalkyl, substituted cycloalkyl, substitutedheterocycloalkyl, substituted aryl, and/or substituted heteroaryl) issubstituted with at least one substituent group, size-limitedsubstituent group, or lower substituent group; wherein if thesubstituted R^(24C) is substituted with a plurality of groups selectedfrom substituent groups, size-limited substituent groups, and lowersubstituent groups; each substituent group, size-limited substituentgroup, and/or lower substituent group may optionally be different. Inembodiments, when R^(24C) is substituted, it is substituted with atleast one substituent group. In embodiments, when R^(24C) issubstituted, it is substituted with at least one size-limitedsubstituent group. In embodiments, when R^(24C) is substituted, it issubstituted with at least one lower substituent group.

In embodiments, a substituted R^(24D) (e.g., substituted alkyl,substituted heteroalkyl, substituted cycloalkyl, substitutedheterocycloalkyl, substituted aryl, and/or substituted heteroaryl) issubstituted with at least one substituent group, size-limitedsubstituent group, or lower substituent group; wherein if thesubstituted R^(24D) is substituted with a plurality of groups selectedfrom substituent groups, size-limited substituent groups, and lowersubstituent groups; each substituent group, size-limited substituentgroup, and/or lower substituent group may optionally be different. Inembodiments, when R^(24D) is substituted, it is substituted with atleast one substituent group. In embodiments, when R^(24D) issubstituted, it is substituted with at least one size-limitedsubstituent group. In embodiments, when R^(24D) is substituted, it issubstituted with at least one lower substituent group.

In embodiments, a substituted L^(9A) (e.g., substituted alkylene,substituted heteroalkylene, substituted cycloalkylene, substitutedheterocycloalkylene, substituted arylene, and/or substitutedheteroarylene) is substituted with at least one substituent group,size-limited substituent group, or lower substituent group; wherein ifthe substituted L^(9A) is substituted with a plurality of groupsselected from substituent groups, size-limited substituent groups, andlower substituent groups; each substituent group, size-limitedsubstituent group, and/or lower substituent group may optionally bedifferent. In embodiments, when L^(9A) is substituted, it is substitutedwith at least one substituent group. In embodiments, when L^(9A) issubstituted, it is substituted with at least one size-limitedsubstituent group. In embodiments, when L^(9A) is substituted, it issubstituted with at least one lower substituent group.

In embodiments, a substituted L^(9B) (e.g., substituted alkylene,substituted heteroalkylene, substituted cycloalkylene, substitutedheterocycloalkylene, substituted arylene, and/or substitutedheteroarylene) is substituted with at least one substituent group,size-limited substituent group, or lower substituent group; wherein ifthe substituted L^(9B) is substituted with a plurality of groupsselected from substituent groups, size-limited substituent groups, andlower substituent groups; each substituent group, size-limitedsubstituent group, and/or lower substituent group may optionally bedifferent. In embodiments, when L^(9B) is substituted, it is substitutedwith at least one substituent group. In embodiments, when L^(9B) issubstituted, it is substituted with at least one size-limitedsubstituent group. In embodiments, when L^(9B) is substituted, it issubstituted with at least one lower substituent group.

In embodiments, a substituted L^(9C) (e.g., substituted alkylene,substituted heteroalkylene, substituted cycloalkylene, substitutedheterocycloalkylene, substituted arylene, and/or substitutedheteroarylene) is substituted with at least one substituent group,size-limited substituent group, or lower substituent group; wherein ifthe substituted L^(9C) is substituted with a plurality of groupsselected from substituent groups, size-limited substituent groups, andlower substituent groups; each substituent group, size-limitedsubstituent group, and/or lower substituent group may optionally bedifferent. In embodiments, when L^(9C) is substituted, it is substitutedwith at least one substituent group. In embodiments, when L^(9C) issubstituted, it is substituted with at least one size-limitedsubstituent group. In embodiments, when L^(9C) is substituted, it issubstituted with at least one lower substituent group.

In embodiments, a substituted L^(9D) (e.g., substituted alkylene,substituted heteroalkylene, substituted cycloalkylene, substitutedheterocycloalkylene, substituted arylene, and/or substitutedheteroarylene) is substituted with at least one substituent group,size-limited substituent group, or lower substituent group; wherein ifthe substituted L^(9D) is substituted with a plurality of groupsselected from substituent groups, size-limited substituent groups, andlower substituent groups; each substituent group, size-limitedsubstituent group, and/or lower substituent group may optionally bedifferent. In embodiments, when L^(9D) is substituted, it is substitutedwith at least one substituent group. In embodiments, when L^(9D) issubstituted, it is substituted with at least one size-limitedsubstituent group. In embodiments, when L^(9D) is substituted, it issubstituted with at least one lower substituent group.

In embodiments, a substituted L^(9E) (e.g., substituted alkylene,substituted heteroalkylene, substituted cycloalkylene, substitutedheterocycloalkylene, substituted arylene, and/or substitutedheteroarylene) is substituted with at least one substituent group,size-limited substituent group, or lower substituent group; wherein ifthe substituted L^(9E) is substituted with a plurality of groupsselected from substituent groups, size-limited substituent groups, andlower substituent groups; each substituent group, size-limitedsubstituent group, and/or lower substituent group may optionally bedifferent. In embodiments, when L^(9E) is substituted, it is substitutedwith at least one substituent group. In embodiments, when L^(9E) issubstituted, it is substituted with at least one size-limitedsubstituent group. In embodiments, when L^(9E) is substituted, it issubstituted with at least one lower substituent group.

In embodiments, the polymeric bioconjugate linker includes a subunithaving the formula:

R¹⁰, R¹², R¹³, and R¹⁴ are independently hydrogen, halogen, —CCl₃,—CBr₃, —CF₃, —CI₃, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I, —CHCl₂, —CHBr₂, —CHF₂,—CHI₂, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂,—NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH,—NHOH, —OCCl₃, —OCBr₃, —OCF₃, —OCI₃, —OCH₂Cl, —OCH₂Br, —OCH₂F, —OCH₂I,—OCHCl₂, —OCHBr₂, —OCHF₂, —OCHI₂, substituted or unsubstituted alkyl(e.g., C₁-C₈, C₁-C₆, C₁-C₄, or C₁-C₂), substituted or unsubstitutedheteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2to 3 membered, or 4 to 5 membered), substituted or unsubstitutedcycloalkyl (e.g., C₃-C₈, C₃-C₆, C₄-C₆, or C₅-C₆), substituted orunsubstituted heterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered,4 to 6 membered, 4 to 5 membered, or 5 to 6 membered), substituted orunsubstituted aryl (e.g., C₆-C₁₀ or phenyl), or substituted orunsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to 9 membered, or 5to 6 membered).

L¹¹ is bonded to the particle. In embodiments, L¹¹ is covalently bondedto the solid surface of the particle. L¹¹ is independently-L^(11A)-L^(11B)-L^(11C)-L^(11D)-L^(11E)-. L^(11A), L^(11B), L^(11C),L^(11D), and L^(11E) are independently a bond, —S(O)₂—, —S(O)—,—S(O)₂NH—, —NH—, —O—, —S—, —SS—, —C(O)—, —C(O)NH—, —C(O)CH₂—, —NHC(O)—,—NHC(O)NH—, —C(O)O—, —OC(O)—, substituted or unsubstituted alkylene(e.g., C₁-C₈, C₁-C₆, C₁-C₄, or C₁-C₂), substituted or unsubstitutedheteroalkylene (e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6 membered,2 to 3 membered, or 4 to 5 membered), substituted or unsubstitutedcycloalkylene (e.g., C₃-C₈, C₃-C₆, C₄-C₆, or C₅-C₆), substituted orunsubstituted heterocycloalkylene (e.g., 3 to 8 membered, 3 to 6membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered),substituted or unsubstituted arylene (e.g., C₆-C₁₀ or phenylene), orsubstituted or unsubstituted heteroarylene (e.g., 5 to 10 membered, 5 to9 membered, or 5 to 6 membered). The symbol z3 is independently aninteger from 1 to 5000.

In embodiments, the polymeric bioconjugate linker is covalently bondedto the solid surface by a linker L¹², wherein L¹² is-L^(12A)-L^(12B)-L^(12C)-L^(12D)-L^(12E)-. L^(12A), L^(12B), L^(12C),L^(12D) and L^(12E) are independently a bond, —S(O)₂—, —S(O)—,—S(O)₂NH—, —NH—, —O—, —S—, —C(O)—, —C(O)NH—, —C(O)CH₂—, —NHC(O)—,—NHC(O)NH—, —C(O)O—, —OC(O)—, substituted or unsubstituted alkylene(e.g., C₁-C₈, C₁-C₆, C₁-C₄, or C₁-C₂), substituted or unsubstitutedheteroalkylene (e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6 membered,2 to 3 membered, or 4 to 5 membered), substituted or unsubstitutedcycloalkylene (e.g., C₃-C₈, C₃-C₆, C₄-C₆, or C₅-C₆), substituted orunsubstituted heterocycloalkylene (e.g., 3 to 8 membered, 3 to 6membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered),substituted or unsubstituted arylene (e.g., C₆-C₁₀ or phenylene), orsubstituted or unsubstituted heteroarylene (e.g., 5 to 10 membered, 5 to9 membered, or 5 to 6 membered).

In embodiments, the polymeric bioconjugate linker does not include L¹²when the polymer includes a subunit of formula (II).

In embodiments, a substituted R¹⁰ (e.g., substituted alkyl, substitutedheteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl,substituted aryl, and/or substituted heteroaryl) is substituted with atleast one substituent group, size-limited substituent group, or lowersubstituent group; wherein if the substituted R¹⁰ is substituted with aplurality of groups selected from substituent groups, size-limitedsubstituent groups, and lower substituent groups; each substituentgroup, size-limited substituent group, and/or lower substituent groupmay optionally be different. In embodiments, when R¹⁰ is substituted, itis substituted with at least one substituent group. In embodiments, whenR¹⁰ is substituted, it is substituted with at least one size-limitedsubstituent group. In embodiments, when R¹⁰ is substituted, it issubstituted with at least one lower substituent group.

In embodiments, a substituted R¹² (e.g., substituted alkyl, substitutedheteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl,substituted aryl, and/or substituted heteroaryl) is substituted with atleast one substituent group, size-limited substituent group, or lowersubstituent group; wherein if the substituted R¹² is substituted with aplurality of groups selected from substituent groups, size-limitedsubstituent groups, and lower substituent groups; each substituentgroup, size-limited substituent group, and/or lower substituent groupmay optionally be different. In embodiments, when R¹² is substituted, itis substituted with at least one substituent group. In embodiments, whenR¹² is substituted, it is substituted with at least one size-limitedsubstituent group. In embodiments, when R¹² is substituted, it issubstituted with at least one lower substituent group.

In embodiments, a substituted R¹³ (e.g., substituted alkyl, substitutedheteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl,substituted aryl, and/or substituted heteroaryl) is substituted with atleast one substituent group, size-limited substituent group, or lowersubstituent group; wherein if the substituted R¹³ is substituted with aplurality of groups selected from substituent groups, size-limitedsubstituent groups, and lower substituent groups; each substituentgroup, size-limited substituent group, and/or lower substituent groupmay optionally be different. In embodiments, when R¹³ is substituted, itis substituted with at least one substituent group. In embodiments, whenR¹³ is substituted, it is substituted with at least one size-limitedsubstituent group. In embodiments, when R¹³ is substituted, it issubstituted with at least one lower substituent group.

In embodiments, a substituted R¹⁴ (e.g., substituted alkyl, substitutedheteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl,substituted aryl, and/or substituted heteroaryl) is substituted with atleast one substituent group, size-limited substituent group, or lowersubstituent group; wherein if the substituted R¹⁴ is substituted with aplurality of groups selected from substituent groups, size-limitedsubstituent groups, and lower substituent groups; each substituentgroup, size-limited substituent group, and/or lower substituent groupmay optionally be different. In embodiments, when R¹⁴ is substituted, itis substituted with at least one substituent group. In embodiments, whenR¹⁴ is substituted, it is substituted with at least one size-limitedsubstituent group. In embodiments, when R¹⁴ is substituted, it issubstituted with at least one lower substituent group.

In embodiments, a substituted L^(11A) (e.g., substituted alkylene,substituted heteroalkylene, substituted cycloalkylene, substitutedheterocycloalkylene, substituted arylene, and/or substitutedheteroarylene) is substituted with at least one substituent group,size-limited substituent group, or lower substituent group; wherein ifthe substituted L^(11A) is substituted with a plurality of groupsselected from substituent groups, size-limited substituent groups, andlower substituent groups; each substituent group, size-limitedsubstituent group, and/or lower substituent group may optionally bedifferent. In embodiments, when L^(11A) is substituted, it issubstituted with at least one substituent group. In embodiments, whenL^(11A) is substituted, it is substituted with at least one size-limitedsubstituent group. In embodiments, when L^(11A) is substituted, it issubstituted with at least one lower substituent group.

In embodiments, a substituted L^(11B) (e.g., substituted alkylene,substituted heteroalkylene, substituted cycloalkylene, substitutedheterocycloalkylene, substituted arylene, and/or substitutedheteroarylene) is substituted with at least one substituent group,size-limited substituent group, or lower substituent group; wherein ifthe substituted L^(11B) is substituted with a plurality of groupsselected from substituent groups, size-limited substituent groups, andlower substituent groups; each substituent group, size-limitedsubstituent group, and/or lower substituent group may optionally bedifferent. In embodiments, when L^(11B) is substituted, it issubstituted with at least one substituent group. In embodiments, whenL^(11B) is substituted, it is substituted with at least one size-limitedsubstituent group. In embodiments, when L^(11B) is substituted, it issubstituted with at least one lower substituent group.

In embodiments, a substituted L^(11C) (e.g., substituted alkylene,substituted heteroalkylene, substituted cycloalkylene, substitutedheterocycloalkylene, substituted arylene, and/or substitutedheteroarylene) is substituted with at least one substituent group,size-limited substituent group, or lower substituent group; wherein ifthe substituted L^(11C) is substituted with a plurality of groupsselected from substituent groups, size-limited substituent groups, andlower substituent groups; each substituent group, size-limitedsubstituent group, and/or lower substituent group may optionally bedifferent. In embodiments, when L^(11C) is substituted, it issubstituted with at least one substituent group. In embodiments, whenL^(11C) is substituted, it is substituted with at least one size-limitedsubstituent group. In embodiments, when L^(11C) is substituted, it issubstituted with at least one lower substituent group.

In embodiments, a substituted L^(11D) (e.g., substituted alkylene,substituted heteroalkylene, substituted cycloalkylene, substitutedheterocycloalkylene, substituted arylene, and/or substitutedheteroarylene) is substituted with at least one substituent group,size-limited substituent group, or lower substituent group; wherein ifthe substituted L^(11D) is substituted with a plurality of groupsselected from substituent groups, size-limited substituent groups, andlower substituent groups; each substituent group, size-limitedsubstituent group, and/or lower substituent group may optionally bedifferent. In embodiments, when L^(11D) is substituted, it issubstituted with at least one substituent group. In embodiments, whenL^(11D) is substituted, it is substituted with at least one size-limitedsubstituent group. In embodiments, when L^(11D) is substituted, it issubstituted with at least one lower substituent group.

In embodiments, a substituted L^(11E) (e.g., substituted alkylene,substituted heteroalkylene, substituted cycloalkylene, substitutedheterocycloalkylene, substituted arylene, and/or substitutedheteroarylene) is substituted with at least one substituent group,size-limited substituent group, or lower substituent group; wherein ifthe substituted L^(11E) is substituted with a plurality of groupsselected from substituent groups, size-limited substituent groups, andlower substituent groups; each substituent group, size-limitedsubstituent group, and/or lower substituent group may optionally bedifferent. In embodiments, when L^(11E) is substituted, it issubstituted with at least one substituent group. In embodiments, whenL^(11E) is substituted, it is substituted with at least one size-limitedsubstituent group. In embodiments, when L^(11E) is substituted, it issubstituted with at least one lower substituent group.

In embodiments, a substituted L^(12A) (e.g., substituted alkylene,substituted heteroalkylene, substituted cycloalkylene, substitutedheterocycloalkylene, substituted arylene, and/or substitutedheteroarylene) is substituted with at least one substituent group,size-limited substituent group, or lower substituent group; wherein ifthe substituted L^(12A) is substituted with a plurality of groupsselected from substituent groups, size-limited substituent groups, andlower substituent groups; each substituent group, size-limitedsubstituent group, and/or lower substituent group may optionally bedifferent. In embodiments, when L^(12A) is substituted, it issubstituted with at least one substituent group. In embodiments, whenL^(12A) is substituted, it is substituted with at least one size-limitedsubstituent group. In embodiments, when L^(12A) is substituted, it issubstituted with at least one lower substituent group.

In embodiments, a substituted L^(12B) (e.g., substituted alkylene,substituted heteroalkylene, substituted cycloalkylene, substitutedheterocycloalkylene, substituted arylene, and/or substitutedheteroarylene) is substituted with at least one substituent group,size-limited substituent group, or lower substituent group; wherein ifthe substituted L^(12B) is substituted with a plurality of groupsselected from substituent groups, size-limited substituent groups, andlower substituent groups; each substituent group, size-limitedsubstituent group, and/or lower substituent group may optionally bedifferent. In embodiments, when L^(12B) is substituted, it issubstituted with at least one substituent group. In embodiments, whenL^(12B) is substituted, it is substituted with at least one size-limitedsubstituent group. In embodiments, when L^(12B) is substituted, it issubstituted with at least one lower substituent group.

In embodiments, a substituted L^(12C) (e.g., substituted alkylene,substituted heteroalkylene, substituted cycloalkylene, substitutedheterocycloalkylene, substituted arylene, and/or substitutedheteroarylene) is substituted with at least one substituent group,size-limited substituent group, or lower substituent group; wherein ifthe substituted L^(12C) is substituted with a plurality of groupsselected from substituent groups, size-limited substituent groups, andlower substituent groups; each substituent group, size-limitedsubstituent group, and/or lower substituent group may optionally bedifferent. In embodiments, when L^(12C) is substituted, it issubstituted with at least one substituent group. In embodiments, whenL^(12C) is substituted, it is substituted with at least one size-limitedsubstituent group. In embodiments, when L^(12C) is substituted, it issubstituted with at least one lower substituent group.

In embodiments, a substituted L^(12D) (e.g., substituted alkylene,substituted heteroalkylene, substituted cycloalkylene, substitutedheterocycloalkylene, substituted arylene, and/or substitutedheteroarylene) is substituted with at least one substituent group,size-limited substituent group, or lower substituent group; wherein ifthe substituted L^(12D) is substituted with a plurality of groupsselected from substituent groups, size-limited substituent groups, andlower substituent groups; each substituent group, size-limitedsubstituent group, and/or lower substituent group may optionally bedifferent. In embodiments, when L^(12D) is substituted, it issubstituted with at least one substituent group. In embodiments, whenL^(12D) is substituted, it is substituted with at least one size-limitedsubstituent group. In embodiments, when L^(12D) is substituted, it issubstituted with at least one lower substituent group.

In embodiments, a substituted L^(12E) (e.g., substituted alkylene,substituted heteroalkylene, substituted cycloalkylene, substitutedheterocycloalkylene, substituted arylene, and/or substitutedheteroarylene) is substituted with at least one substituent group,size-limited substituent group, or lower substituent group; wherein ifthe substituted L^(12E) is substituted with a plurality of groupsselected from substituent groups, size-limited substituent groups, andlower substituent groups; each substituent group, size-limitedsubstituent group, and/or lower substituent group may optionally bedifferent. In embodiments, when L^(12E) is substituted, it issubstituted with at least one substituent group. In embodiments, whenL^(12E) is substituted, it is substituted with at least one size-limitedsubstituent group. In embodiments, when L^(12E) is substituted, it issubstituted with at least one lower substituent group.

In embodiments, the polymeric bioconjugate linker includes a subunithaving the formula.

-   -   R¹⁵, R¹⁷, R⁸, and R¹⁹ are independently hydrogen, halogen,        —CCl₃, —CBr₃, —CF₃, —CI₃, —CH₂Cl, —CH₂Br, —CH₂F, —CH₂I, —CHCl₂,        —CHBr₂, —CHF₂, —CHI₂, —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH,        —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂,        —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃, —OCBr₃, —OCF₃,        —OCI₃, —OCH₂Cl, —OCH₂Br, —OCH₂F, —OCH₂I, —OCHCl₂, —OCHBr₂,        —OCHF₂, —OCHI₂, substituted or unsubstituted alkyl (e.g., C₁-C₈,        C₁-C₆, C₁-C₄, or C₁-C₂), substituted or unsubstituted        heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6        membered, 2 to 3 membered, or 4 to 5 membered), substituted or        unsubstituted cycloalkyl (e.g., C₃-C₈, C₃-C₆, C₄-C₆, or C₅-C₆),        substituted or unsubstituted heterocycloalkyl (e.g., 3 to 8        membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or        5 to 6 membered), substituted or unsubstituted aryl (e.g.,        C₆-C₁₀ or phenyl), or substituted or unsubstituted heteroaryl        (e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered).

L¹⁶ is independently -L^(16A)-L^(16B)-L^(16C)-L^(16D)-L^(16E)-. L^(16A),L^(16B) L^(16C), L^(16D), and L^(16E) are independently a bond, —S(O)₂—,—S(O)—, —S(O)₂NH—, —NH—, —O—, —S—, —C(O)—, —C(O)NH—, —C(O)CH₂—,—NHC(O)—, —NHC(O)NH—, —C(O)O—, —OC(O)—, substituted or unsubstitutedalkylene (e.g., C₁-C₈, C₁-C₆, C₁-C₄, or C₁-C₂), substituted orunsubstituted heteroalkylene (e.g., 2 to 8 membered, 2 to 6 membered, 4to 6 membered, 2 to 3 membered, or 4 to 5 membered), substituted orunsubstituted cycloalkylene (e.g., C₃-C₈, C₃-C₆, C₄-C₆, or C₅-C₆),substituted or unsubstituted heterocycloalkylene (e.g., 3 to 8 membered,3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered),substituted or unsubstituted arylene (e.g., C₆-C₁₀ or phenylene), orsubstituted or unsubstituted heteroarylene (e.g., 5 to 10 membered, 5 to9 membered, or 5 to 6 membered).

R¹⁶ is independently hydrogen, halogen, —CX¹⁶ ₃, —CHX¹⁶ ₂, —CH₂X¹⁶,—OCX¹⁶ ₃, —OCH₂X¹⁶, —OCHX¹⁶ ₂, —CN, —SO_(n16)R^(16D),—SO_(v16)NR^(16A)R^(16B), —NHC(O)NR^(16A)R^(16B), —N(O)_(m16),—NR^(16A)R^(16B), —C(O)R^(16C), —C(O)—OR^(16C), —C(O)NR^(16A)R^(16B),—OR^(16D), —NR^(16A)SO₂R^(16D), —NR^(16A)C(O)R^(16C),—NR^(16A)C(O)OR^(16C), —NR^(16A)OR^(16C), —OC(O)R^(16C), substituted orunsubstituted alkyl (e.g., C₁-C₈, C₁-C₆, C₁-C₄, or C₁-C₂), substitutedor unsubstituted heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered, 4to 6 membered, 2 to 3 membered, or 4 to 5 membered), substituted orunsubstituted cycloalkyl (e.g., C₃-C₈, C₃-C₆, C₄-C₆, or C₅-C₆),substituted or unsubstituted heterocycloalkyl (e.g., 3 to 8 membered, 3to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered),substituted or unsubstituted aryl (e.g., C₆-C₁₀ or phenyl), orsubstituted or unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to 9membered, or 5 to 6 membered); wherein R¹⁶ is a third non-reactivemoiety. R^(16A), R^(16B), R^(16C), and R^(16D) are independentlyhydrogen, —CX₃, —CN, —COOH, —CONH₂, —CHX₂, —CH₂X, substituted orunsubstituted alkyl (e.g., C₁-C₈, C₁-C₆, C₁-C₄, or C₁-C₂), substitutedor unsubstituted heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered, 4to 6 membered, 2 to 3 membered, or 4 to 5 membered), substituted orunsubstituted cycloalkyl (e.g., C₃-C₈, C₃-C₆, C₄-C₆, or C₅-C₆),substituted or unsubstituted heterocycloalkyl (e.g., 3 to 8 membered, 3to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered),substituted or unsubstituted aryl (e.g., C₆-C₁₀ or phenyl), substitutedor unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to 9 membered, or5 to 6 membered), a protecting group, or a leaving group; R^(16A) andR^(16B) substituents bonded to the same nitrogen atom may optionally bejoined to form a substituted or unsubstituted heterocycloalkyl (e.g., 3to 8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5to 6 membered) or substituted or unsubstituted heteroaryl (e.g., 5 to 10membered, 5 to 9 membered, or 5 to 6 membered).

The symbol z4 is independently an integer from 1 to 5000. X and X¹⁶ areindependently —F, —Cl, —Br, or —I. The symbol n16 is independently aninteger from 0 to 4. The symbols v16 and m16 are each independently 1 to2.

In embodiments, a substituted R¹⁵ (e.g., substituted alkyl, substitutedheteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl,substituted aryl, and/or substituted heteroaryl) is substituted with atleast one substituent group, size-limited substituent group, or lowersubstituent group; wherein if the substituted R¹⁵ is substituted with aplurality of groups selected from substituent groups, size-limitedsubstituent groups, and lower substituent groups; each substituentgroup, size-limited substituent group, and/or lower substituent groupmay optionally be different. In embodiments, when R¹⁵ is substituted, itis substituted with at least one substituent group. In embodiments, whenR¹⁵ is substituted, it is substituted with at least one size-limitedsubstituent group. In embodiments, when R¹⁵ is substituted, it issubstituted with at least one lower substituent group.

In embodiments, a substituted R¹⁷ (e.g., substituted alkyl, substitutedheteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl,substituted aryl, and/or substituted heteroaryl) is substituted with atleast one substituent group, size-limited substituent group, or lowersubstituent group; wherein if the substituted R¹⁷ is substituted with aplurality of groups selected from substituent groups, size-limitedsubstituent groups, and lower substituent groups; each substituentgroup, size-limited substituent group, and/or lower substituent groupmay optionally be different. In embodiments, when R¹⁷ is substituted, itis substituted with at least one substituent group. In embodiments, whenR¹⁷ is substituted, it is substituted with at least one size-limitedsubstituent group. In embodiments, when R¹⁷ is substituted, it issubstituted with at least one lower substituent group.

In embodiments, a substituted R¹⁸ (e.g., substituted alkyl, substitutedheteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl,substituted aryl, and/or substituted heteroaryl) is substituted with atleast one substituent group, size-limited substituent group, or lowersubstituent group; wherein if the substituted R¹⁸ is substituted with aplurality of groups selected from substituent groups, size-limitedsubstituent groups, and lower substituent groups; each substituentgroup, size-limited substituent group, and/or lower substituent groupmay optionally be different. In embodiments, when R¹⁸ is substituted, itis substituted with at least one substituent group. In embodiments, whenR¹⁸ is substituted, it is substituted with at least one size-limitedsubstituent group. In embodiments, when R¹⁸ is substituted, it issubstituted with at least one lower substituent group.

In embodiments, a substituted R¹⁹ (e.g., substituted alkyl, substitutedheteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl,substituted aryl, and/or substituted heteroaryl) is substituted with atleast one substituent group, size-limited substituent group, or lowersubstituent group; wherein if the substituted R¹⁹ is substituted with aplurality of groups selected from substituent groups, size-limitedsubstituent groups, and lower substituent groups; each substituentgroup, size-limited substituent group, and/or lower substituent groupmay optionally be different. In embodiments, when R¹⁹ is substituted, itis substituted with at least one substituent group. In embodiments, whenR¹⁹ is substituted, it is substituted with at least one size-limitedsubstituent group. In embodiments, when R¹⁹ is substituted, it issubstituted with at least one lower substituent group.

In embodiments, a substituted L^(16A) (e.g., substituted alkylene,substituted heteroalkylene, substituted cycloalkylene, substitutedheterocycloalkylene, substituted arylene, and/or substitutedheteroarylene) is substituted with at least one substituent group,size-limited substituent group, or lower substituent group; wherein ifthe substituted L^(16A) is substituted with a plurality of groupsselected from substituent groups, size-limited substituent groups, andlower substituent groups; each substituent group, size-limitedsubstituent group, and/or lower substituent group may optionally bedifferent. In embodiments, when L^(16A) is substituted, it issubstituted with at least one substituent group. In embodiments, whenL^(16A) is substituted, it is substituted with at least one size-limitedsubstituent group. In embodiments, when L^(16A) is substituted, it issubstituted with at least one lower substituent group.

In embodiments, a substituted L^(16B) (e.g., substituted alkylene,substituted heteroalkylene, substituted cycloalkylene, substitutedheterocycloalkylene, substituted arylene, and/or substitutedheteroarylene) is substituted with at least one substituent group,size-limited substituent group, or lower substituent group; wherein ifthe substituted L^(16B) is substituted with a plurality of groupsselected from substituent groups, size-limited substituent groups, andlower substituent groups; each substituent group, size-limitedsubstituent group, and/or lower substituent group may optionally bedifferent. In embodiments, when L^(16B) is substituted, it issubstituted with at least one substituent group. In embodiments, whenL^(16B) is substituted, it is substituted with at least one size-limitedsubstituent group. In embodiments, when L^(16B) is substituted, it issubstituted with at least one lower substituent group.

In embodiments, a substituted L^(16C) (e.g., substituted alkylene,substituted heteroalkylene, substituted cycloalkylene, substitutedheterocycloalkylene, substituted arylene, and/or substitutedheteroarylene) is substituted with at least one substituent group,size-limited substituent group, or lower substituent group; wherein ifthe substituted L^(16C) is substituted with a plurality of groupsselected from substituent groups, size-limited substituent groups, andlower substituent groups; each substituent group, size-limitedsubstituent group, and/or lower substituent group may optionally bedifferent. In embodiments, when L^(16C) is substituted, it issubstituted with at least one substituent group. In embodiments, whenL^(16C) is substituted, it is substituted with at least one size-limitedsubstituent group. In embodiments, when L^(16C) is substituted, it issubstituted with at least one lower substituent group.

In embodiments, a substituted L^(16D) (e.g., substituted alkylene,substituted heteroalkylene, substituted cycloalkylene, substitutedheterocycloalkylene, substituted arylene, and/or substitutedheteroarylene) is substituted with at least one substituent group,size-limited substituent group, or lower substituent group; wherein ifthe substituted L^(16D) is substituted with a plurality of groupsselected from substituent groups, size-limited substituent groups, andlower substituent groups; each substituent group, size-limitedsubstituent group, and/or lower substituent group may optionally bedifferent. In embodiments, when L^(16D) is substituted, it issubstituted with at least one substituent group. In embodiments, whenL^(16D) is substituted, it is substituted with at least one size-limitedsubstituent group. In embodiments, when L^(16D) is substituted, it issubstituted with at least one lower substituent group.

In embodiments, a substituted L^(16E) (e.g., substituted alkylene,substituted heteroalkylene, substituted cycloalkylene, substitutedheterocycloalkylene, substituted arylene, and/or substitutedheteroarylene) is substituted with at least one substituent group,size-limited substituent group, or lower substituent group; wherein ifthe substituted L^(16E) is substituted with a plurality of groupsselected from substituent groups, size-limited substituent groups, andlower substituent groups; each substituent group, size-limitedsubstituent group, and/or lower substituent group may optionally bedifferent. In embodiments, when L^(16E) is substituted, it issubstituted with at least one substituent group. In embodiments, whenL^(16E) is substituted, it is substituted with at least one size-limitedsubstituent group. In embodiments, when L^(16E) is substituted, it issubstituted with at least one lower substituent group.

In embodiments, a substituted R¹⁶ (e.g., substituted alkyl, substitutedheteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl,substituted aryl, and/or substituted heteroaryl) is substituted with atleast one substituent group, size-limited substituent group, or lowersubstituent group; wherein if the substituted R¹⁶ is substituted with aplurality of groups selected from substituent groups, size-limitedsubstituent groups, and lower substituent groups; each substituentgroup, size-limited substituent group, and/or lower substituent groupmay optionally be different. In embodiments, when R¹⁶ is substituted, itis substituted with at least one substituent group. In embodiments, whenR¹⁶ is substituted, it is substituted with at least one size-limitedsubstituent group. In embodiments, when R¹⁶ is substituted, it issubstituted with at least one lower substituent group.

In embodiments, a substituted R^(16A) (e.g., substituted alkyl,substituted heteroalkyl, substituted cycloalkyl, substitutedheterocycloalkyl, substituted aryl, and/or substituted heteroaryl) issubstituted with at least one substituent group, size-limitedsubstituent group, or lower substituent group; wherein if thesubstituted R^(16A) is substituted with a plurality of groups selectedfrom substituent groups, size-limited substituent groups, and lowersubstituent groups; each substituent group, size-limited substituentgroup, and/or lower substituent group may optionally be different. Inembodiments, when R^(16A) is substituted, it is substituted with atleast one substituent group. In embodiments, when R^(16A) issubstituted, it is substituted with at least one size-limitedsubstituent group. In embodiments, when R^(16A) is substituted, it issubstituted with at least one lower substituent group.

In embodiments, a substituted R^(16B) (e.g., substituted alkyl,substituted heteroalkyl, substituted cycloalkyl, substitutedheterocycloalkyl, substituted aryl, and/or substituted heteroaryl) issubstituted with at least one substituent group, size-limitedsubstituent group, or lower substituent group; wherein if thesubstituted R^(16B) is substituted with a plurality of groups selectedfrom substituent groups, size-limited substituent groups, and lowersubstituent groups; each substituent group, size-limited substituentgroup, and/or lower substituent group may optionally be different. Inembodiments, when R^(16B) is substituted, it is substituted with atleast one substituent group. In embodiments, when R^(16B) issubstituted, it is substituted with at least one size-limitedsubstituent group. In embodiments, when R^(16B) is substituted, it issubstituted with at least one lower substituent group.

In embodiments, a substituted ring formed when R^(16A) and R^(16B)substituents bonded to the same nitrogen atom are joined (e.g.,substituted heterocycloalkyl and/or substituted heteroaryl) issubstituted with at least one substituent group, size-limitedsubstituent group, or lower substituent group; wherein if thesubstituted ring formed when R^(16A) and R^(16B) substituents bonded tothe same nitrogen atom are joined is substituted with a plurality ofgroups selected from substituent groups, size-limited substituentgroups, and lower substituent groups; each substituent group,size-limited substituent group, and/or lower substituent group mayoptionally be different. In embodiments, when the substituted ringformed when R^(16A) and R^(16B) substituents bonded to the same nitrogenatom are joined is substituted, it is substituted with at least onesubstituent group. In embodiments, when the substituted ring formed whenR^(16A) and R^(16B) substituents bonded to the same nitrogen atom arejoined is substituted, it is substituted with at least one size-limitedsubstituent group. In embodiments, when the substituted ring formed whenR^(16A) and R^(16B) substituents bonded to the same nitrogen atom arejoined is substituted, it is substituted with at least one lowersubstituent group.

In embodiments, a substituted R^(16C) (e.g., substituted alkyl,substituted heteroalkyl, substituted cycloalkyl, substitutedheterocycloalkyl, substituted aryl, and/or substituted heteroaryl) issubstituted with at least one substituent group, size-limitedsubstituent group, or lower substituent group; wherein if thesubstituted R^(16C) is substituted with a plurality of groups selectedfrom substituent groups, size-limited substituent groups, and lowersubstituent groups; each substituent group, size-limited substituentgroup, and/or lower substituent group may optionally be different. Inembodiments, when R^(16C) is substituted, it is substituted with atleast one substituent group. In embodiments, when R^(16C) issubstituted, it is substituted with at least one size-limitedsubstituent group. In embodiments, when R^(16C) is substituted, it issubstituted with at least one lower substituent group.

In embodiments, a substituted R^(16D) (e.g., substituted alkyl,substituted heteroalkyl, substituted cycloalkyl, substitutedheterocycloalkyl, substituted aryl, and/or substituted heteroaryl) issubstituted with at least one substituent group, size-limitedsubstituent group, or lower substituent group; wherein if thesubstituted R^(16D) is substituted with a plurality of groups selectedfrom substituent groups, size-limited substituent groups, and lowersubstituent groups; each substituent group, size-limited substituentgroup, and/or lower substituent group may optionally be different. Inembodiments, when R^(16D) is substituted, it is substituted with atleast one substituent group. In embodiments, when R^(16D) issubstituted, it is substituted with at least one size-limitedsubstituent group. In embodiments, when R^(16D) is substituted, it issubstituted with at least one lower substituent group.

In embodiments, R²¹ is independently —CN. In embodiments, R²¹ isindependently substituted or unsubstituted alkyl. In embodiments, R²¹ isindependently substituted or unsubstituted C₁-C₄ alkyl. In embodiments,R²¹ is independently unsubstituted C₁-C₄ alkyl. In embodiments, R²¹ isindependently unsubstituted methyl. In embodiments, R²¹ is independentlyunsubstituted ethyl. In embodiments, R²¹ is independently unsubstitutedpropyl. In embodiments, R²¹ is independently unsubstituted n-propyl. Inembodiments, R²¹ is independently unsubstituted isopropyl. Inembodiments, R²¹ is independently unsubstituted butyl. In embodiments,R²¹ is independently unsubstituted n-butyl. In embodiments, R²¹ isindependently unsubstituted tert-butyl. In embodiments, R²¹ isindependently substituted or unsubstituted heteroalkyl. In embodiments,R²¹ is independently substituted or unsubstituted 2 to 6 memberedheteroalkyl. In embodiments, each R²¹ is the same.

In embodiments, L² is independently a substituted or unsubstitutedheteroalkylene. In embodiments, L^(2A) is independently a bond. Inembodiments, L^(2B) and L^(2D) are independently substituted orunsubstituted heteroalkylene; L^(2C) is independently a substituted orunsubstituted heteroarylene; and L^(2E) is independently a bond. Inembodiments, L^(2B) and L^(2E) are independently substituted orunsubstituted heteroalkylene; L^(2C) is independently a substituted orunsubstituted heteroarylene; and L^(2D) is independently a substitutedor unsubstituted arylene.

In embodiments, L^(2A) is independently a substituted or unsubstitutedheteroalkylene. In embodiments, L^(2A) is independently

In embodiments, L^(2A) is independently

In embodiments, L^(2A) is independently

In embodiments, L^(2A) is independently

In embodiments, L^(2A) is independently

In embodiments, L^(2A) is independently

In embodiments, L^(2A) is independently

In embodiments, L^(2A) is independently

In embodiments, L^(2A) is independently

In embodiments, L^(2A) is independently

In embodiments, L^(2A) is independently

In embodiments, L^(2A) is independently

In embodiments, L^(2A) is independently

In embodiments, L^(2A) is independently

In embodiments, L^(2A) is independently

In embodiments, L^(2A) is independently

In embodiments, L^(2B) is independently a substituted or unsubstitutedheteroarylene. In embodiments, L^(2B) is independently

In embodiments, L^(2B) is independently

In embodiments, L^(2B) is independently

In embodiments, L^(2B) is independently

In embodiments, L^(2B) is independently

In embodiments, L^(2B) is independently

In embodiments, L^(2B) is independently

In embodiments, L^(2B) is independently

In embodiments, L^(2B) is independently

In embodiments, L^(2C) is independently a substituted or unsubstitutedheteroalkylene. In embodiments, L^(2C) is independently

wherein n is independently an integer from 4 to 12. In embodiments,L^(2C) is independently

wherein n is independently an integer from 4 to 12 and m isindependently an integer from 4 to 12. In embodiments, L^(2C) isindependently

wherein n is independently an integer from 4 to 12. In embodiments,L^(2C) is independently

wherein n is independently an integer from 4 to 12. In embodiments,L^(2C) is independently

wherein n is independently an integer from 4 to 12 and m isindependently an integer from 4 to 12. In embodiments, L^(2C) isindependently

wherein n is independently an integer from 4 to 12 and m isindependently an integer from 4 to 12.

In embodiments, L^(2D) is independently a substituted or unsubstitutedheteroalkylene. In embodiments, L^(2D) is independently

wherein n is independently an integer from 4 to 12. In embodiments,L^(2D) is independently

wherein n is independently an integer from 4 to 12 and m isindependently an integer from 4 to 12. In embodiments, L^(2D) isindependently

wherein n is independently an integer from 4 to 12. In embodiments,L^(2D) is independently

wherein n is independently an integer from 4 to 12. In embodiments,L^(2D) is independently

wherein n is independently an integer from 4 to 12 and m isindependently an integer from 4 to 12. In embodiments, L^(2D) isindependently

wherein n is independently an integer from 4 to 12 and m isindependently an integer from 4 to 12.

In embodiments, -L^(2C)-L^(2D)-L^(2E)_is independently a substituted orunsubstituted heteroalkylene. In embodiments, -L^(2C)-L^(2D)-L^(2E)_isindependently

wherein n is independently an integer from 4 to 12. In embodiments,-L^(2C)-L^(2D)-L^(2E)- is independently

wherein n is independently an integer from 4 to 12 and m isindependently an integer from 4 to 12. In embodiments,-L^(2C)-L^(2D)-L^(2E)_is independently

wherein n is independently an integer from 4 to 12. In embodiments,-L^(2C)-L^(2D)-L^(2E)- is independently

wherein n is independently an integer from 4 to 12. In embodiments,-L^(2C)-L^(2D)-L^(2E)- is independently

wherein n is independently an integer from 4 to 12 and m isindependently an integer from 4 to 12. In embodiments,-L^(2C)-L^(2D)-L^(2E)- is independently

wherein n is independently an integer from 4 to 12 and m isindependently an integer from 4 to 12.

In embodiments, L² is independently

wherein L^(2A), L^(2C), L^(2D), and L^(2E) are as described herein,including in embodiments.

In embodiments, -L^(2A)-L^(2B)-L^(2C)- is independently

wherein n is independently an integer from 4 to 12. In embodiments,-L^(2A)-L^(2B)-L^(2C)- is independently

wherein n is independently an integer from 4 to 12. In embodiments,-L^(2A)-L^(2B)-L^(2C)- is independently

wherein n is independently an integer from 4 to 12. In embodiments,-L^(2A)-L^(2B)-L^(2C)- is independently

wherein n is independently an integer from 4 to 12. In embodiments,-L^(2A)-L^(2B)-L^(2C)- is independently

wherein n is independently an integer from 4 to 12. In embodiments,-L^(2A)-L^(2B)-L^(2C)- is independently

wherein n is independently an integer from 4 to 12. In embodiments,-L^(2A)-L^(2B)-L^(2C)- is independently

wherein n is independently an integer from 4 to 12. In embodiments,-L^(2A)-L^(2B)-L^(2C)- is independently

wherein n is independently an integer from 4 to 12. In embodiments,-L^(2A)-L^(2B)-L^(2C)- is independently

wherein n is independently an integer from 4 to 12. In embodiments,-L^(2A)-L^(2B)-L^(2C)- is independently

wherein n is independently an integer from 4 to 12. In embodiments,-L^(2A)-L^(2B)-L^(2C)- is independently

wherein n is independently an integer from 4 to 12. In embodiments,-L^(2A)-L^(2B)-L^(2C)- is independently

wherein n is independently an integer from 4 to 12. In embodiments,-L^(2A)-L^(2B)-L^(2C)- is independently

wherein n is independently an integer from 4 to 12. In embodiments,-L^(2A)-L^(2B)-L^(2C)- is independently

wherein n is independently an integer from 4 to 12. In embodiments,-L^(2A)-L^(2B)-L^(2C)- is independently

wherein n is independently an integer from 4 to 12. In embodiments,-L^(2A)-L^(2B)-L^(2C)- is independently

wherein n is independently an integer from 4 to 12.

In embodiments, L^(2D) is independently —S—S— and L^(2E) isindependently an unsubstituted C₄-C₈ alkylene. In embodiments, L^(2D) isindependently

and L^(2E) is independently an unsubstituted C₄-C₈ alkylene.

In embodiments, L² is independently a substituted or unsubstitutedheteroalkylene. In embodiments, L² is independently

The symbol n is independently an integer from 4 to 12, and the symbol mis an integer from 4 to 12. In embodiments, L² is independently

The symbol n is independently an integer from 4 to 12, and the symbol mis independently an integer from 4 to 12. In embodiments, L² isindependently

The symbol n is independently an integer from 4 to 12, and the symbol mis independently an integer from 4 to 12. In embodiments, L² isindependently

The symbol n is independently an integer from 4 to 12, and the symbol mis independently an integer from 4 to 12. In embodiments, each L² is thesame.

In embodiments, R²³ is independently —CN. In embodiments, R²³ isindependently substituted or unsubstituted alkyl. In embodiments, R²³ isindependently substituted or unsubstituted C₁-C₄ alkyl. In embodiments,R²³ is independently unsubstituted C₁-C₄ alkyl. In embodiments, R²³ isindependently unsubstituted methyl. In embodiments, R²³ is independentlyunsubstituted ethyl. In embodiments, R²³ is independently unsubstitutedpropyl. In embodiments, R²³ is independently unsubstituted n-propyl. Inembodiments, R²³ is independently unsubstituted isopropyl. Inembodiments, R²³ is independently unsubstituted butyl. In embodiments,R²³ is independently unsubstituted n-butyl. In embodiments, R²³ isindependently unsubstituted tert-butyl. In embodiments, R²³ isindependently substituted or unsubstituted heteroalkyl. In embodiments,R²³ is independently substituted or unsubstituted 2 to 6 memberedheteroalkyl. In embodiments, each R²³ is the same.

In embodiments, L⁴ is independently substituted or unsubstitutedheteroalkylene. In embodiments, L^(4B) and L^(4D) are independentlysubstituted or unsubstituted heteroalkylene; L^(4C) is independently asubstituted or unsubstituted heteroarylene; and L^(4E) is independentlya bond. In embodiments, L^(4B) and L^(4E) are independently substitutedor unsubstituted heteroalkylene; L^(4C) is independently a substitutedor unsubstituted heteroarylene; and L^(4D) is independently asubstituted or unsubstituted arylene. In embodiments, L^(4A) isindependently a bond. In embodiments, each L⁴ is the same.

In embodiments, R²⁴ is independently a non-reactive moiety selected fromhydrogen, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CH₂Cl, —CH₂Br, —CH₂F,—CH₂I, —CHCl₂, —CHBr₂, —CHF₂, —CHI₂, —CN, —OH, —NH₂, —COOH, —CONH₂,—NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂,—NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃, —OCBr₃, —OCF₃,—OCI₃, —OCH₂Cl, —OCH₂Br, —OCH₂F, —OCH₂I, —OCHCl₂, —OCHBr₂, —OCHF₂,—OCHI₂, substituted or unsubstituted alkyl (e.g., C₁-C₈, C₁-C₆, C₁-C₄,or C₁-C₂), substituted or unsubstituted heteroalkyl (e.g., 2 to 8membered, 2 to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4 to 5membered), substituted or unsubstituted cycloalkyl (e.g., C₃-C₈, C₃-C₆,C₄-C₆, or C₅-C₆), substituted or unsubstituted heterocycloalkyl (e.g., 3to 8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5to 6 membered), substituted or unsubstituted aryl (e.g., C₆-C₁₀ orphenyl), or substituted or unsubstituted heteroaryl (e.g., 5 to 10membered, 5 to 9 membered, or 5 to 6 membered). In embodiments, R²⁴ isindependently a non-reactive moiety selected from hydrogen, substitutedor unsubstituted alkyl (e.g., C₁-C₈, C₁-C₆, C₁-C₄, or C₁-C₂),substituted or unsubstituted heteroalkyl (e.g., 2 to 8 membered, 2 to 6membered, 4 to 6 membered, 2 to 3 membered, or 4 to 5 membered),substituted or unsubstituted cycloalkyl (e.g., C₃-C₈, C₃-C₆, C₄-C₆, orC₅-C₆), substituted or unsubstituted heterocycloalkyl (e.g., 3 to 8membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6membered), substituted or unsubstituted aryl (e.g., C₆-C₁₀ or phenyl),or substituted or unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to9 membered, or 5 to 6 membered). In embodiments, R²⁴ is independentlyhydrogen, —OCH₂CH₂—(OCH₂CH₂)_(p)—OCH₃ or —N(CH₃)₂; and p is an integerfrom 0 to 10. In embodiments, each R²⁴ is the same. In embodiments,-L⁴-R²⁴ is independently —OCH₂CH₂—(OCH₂CH₂)_(p)—OCH₃ or —N(CH₃)₂; and pis an integer from 4 to 10.

In embodiments, R⁵ and R⁶ are independently hydrogen. In embodiments, R⁵is independently hydrogen. In embodiments, R⁵ is independentlysubstituted or unsubstituted C₁-C₄ alkyl. In embodiments, R⁵ isindependently unsubstituted C₁-C₄ alkyl. In embodiments, R⁵ isindependently unsubstituted methyl. In embodiments, R⁵ is independentlyunsubstituted ethyl. In embodiments, R⁵ is independently unsubstitutedpropyl. In embodiments, R⁵ is independently unsubstituted n-propyl. Inembodiments, R⁵ is independently unsubstituted isopropyl. Inembodiments, R⁵ is independently unsubstituted butyl. In embodiments, R⁵is independently unsubstituted n-butyl. In embodiments, R⁵ isindependently unsubstituted tert-butyl. In embodiments, each R⁵ is thesame.

In embodiments, R⁶ is independently hydrogen. In embodiments, R⁶ isindependently substituted or unsubstituted C₁-C₄ alkyl. In embodiments,R⁶ is independently unsubstituted C₁-C₄ alkyl. In embodiments, R⁶ isindependently unsubstituted methyl. In embodiments, R⁶ is independentlyunsubstituted ethyl. In embodiments, R⁶ is independently unsubstitutedpropyl. In embodiments, R⁶ is independently unsubstituted n-propyl. Inembodiments, R⁶ is independently unsubstituted isopropyl. Inembodiments, R⁶ is independently unsubstituted butyl. In embodiments, R⁶is independently unsubstituted n-butyl. In embodiments, R⁶ isindependently unsubstituted tert-butyl. In embodiments, each R⁶ is thesame.

In embodiments, R⁷ and R⁸ are independently hydrogen. In embodiments, R⁷is independently hydrogen. In embodiments, R⁷ is independentlysubstituted or unsubstituted C₁-C₄ alkyl. In embodiments, R⁷ isindependently unsubstituted C₁-C₄ alkyl. In embodiments, R⁷ isindependently unsubstituted methyl. In embodiments, R⁷ is independentlyunsubstituted ethyl. In embodiments, R⁷ is independently unsubstitutedpropyl. In embodiments, R⁷ is independently unsubstituted n-propyl. Inembodiments, R⁷ is independently unsubstituted isopropyl. Inembodiments, R⁷ is independently unsubstituted butyl. In embodiments, R⁷is independently unsubstituted n-butyl. In embodiments, R⁷ isindependently unsubstituted tert-butyl. In embodiments, each R⁷ is thesame.

In embodiments, R⁸ is independently hydrogen. In embodiments, R⁸ isindependently substituted or unsubstituted C₁-C₄ alkyl. In embodiments,R⁸ is independently unsubstituted C₁-C₄ alkyl. In embodiments, R⁸ isindependently unsubstituted methyl. In embodiments, R⁸ is independentlyunsubstituted ethyl. In embodiments, R⁸ is independently unsubstitutedpropyl. In embodiments, R⁸ is independently unsubstituted n-propyl. Inembodiments, R⁸ is independently unsubstituted isopropyl. Inembodiments, R⁸ is independently unsubstituted butyl. In embodiments, R⁸is independently unsubstituted n-butyl. In embodiments, R⁸ isindependently unsubstituted tert-butyl. In embodiments, each R⁸ is thesame.

In embodiments, L^(9A) is independently a bond. In embodiments, L^(9B)and L^(9D) are independently substituted or unsubstitutedheteroalkylene; L^(9C) is independently a substituted or unsubstitutedheteroarylene; and L^(9E) is independently a bond. In embodiments,L^(9B) and L^(9E) are independently substituted or unsubstitutedheteroalkylene; L^(9C) is independently a substituted or unsubstitutedheteroarylene; and L^(9D) is independently a substituted orunsubstituted arylene.

In embodiments, L^(9A) is independently a substituted or unsubstitutedheteroalkylene. In embodiments, L^(9A) is independently

In embodiments, L^(9A) is independently

In embodiments, L^(9A) is independently

In embodiments, L^(A) is independently

In embodiments, L^(9A) is independently

In embodiments, L^(9A) is independently

In embodiments, L^(9A) is independently

In embodiments, L^(9A) is independently

In embodiments, L^(9A) is independently

In embodiments, L^(9A) is independently

In embodiments, L^(9A) is independently

In embodiments, L^(9A) is independently

In embodiments, L^(9A) is independently

In embodiments, L^(9A) is independently

In embodiments, L^(9A) is independently

In embodiments, L^(9A) is independently

In embodiments, L^(9B) is independently a substituted or unsubstitutedheteroarylene. In embodiments, L^(9B) is independently

In embodiments, L^(9B) is independently

In embodiments, L^(9B) is independently

In embodiments, L^(9B) is independently

In embodiments, L^(9B) is independently

In embodiments, L^(9B) is independently

In embodiments, L^(9B) is independently

In embodiments, L^(9B) is independently

In embodiments, L^(9B) is independently

In embodiments, L^(9C) is independently a substituted or unsubstitutedheteroalkylene. In embodiments, L^(9C) is independently

wherein n is independently an integer from 4 to 12. In embodiments,L^(9C) is independently

wherein n is independently an integer from 4 to 12 and m isindependently an integer from 4 to 12. In embodiments, L^(9C) isindependently

wherein n is independently an integer from 4 to 12. In embodiments,L^(9C) is independently

wherein n is independently an integer from 4 to 12. In embodiments,L^(9C) is independently

wherein n is independently an integer from 4 to 12 and m isindependently an integer from 4 to 12. In embodiments, L^(9C) isindependently

wherein n is independently an integer from 4 to 12 and m isindependently an integer from 4 to 12.

In embodiments, L^(9D) is independently a substituted or unsubstitutedheteroalkylene. In embodiments, L^(9D) is independently

wherein n is independently an integer from 4 to 12. In embodiments,L^(9D) is independently

wherein n is independently an integer from 4 to 12 and m isindependently an integer from 4 to 12. In embodiments, L^(9D) isindependently

wherein n is independently an integer from 4 to 12. In embodiments,L^(9D) is independently

wherein n is independently an integer from 4 to 12. In embodiments,L^(9D) is independently

wherein n is independently an integer from 4 to 12 and m isindependently an integer from 4 to 12. In embodiments, L^(9D) isindependently

wherein n is independently an integer from 4 to 12 and m isindependently an integer from 4 to 12.

In embodiments, -L^(9C)-L^(9D)-L^(9E)_is independently a substituted orunsubstituted heteroalkylene. In embodiments, -L^(9C)-L^(9D)-L^(9E)_isindependently

wherein n is independently an integer from 4 to 12. In embodiments,-L^(9C)-L^(9D)-L^(9E)_is independently

wherein n is independently an integer from 4 to 12 and m isindependently an integer from 4 to 12. In embodiments,-L^(9C)-L^(9D)-L^(9E)_is independently

wherein n is independently an integer from 4 to 12. In embodiments,-L^(9C)-L^(9D)-L^(9E)- is independently

wherein n is independently an integer from 4 to 12. In embodiments,-L^(9C)-L^(9D)-L^(9E)- is independently

wherein n is independently an integer from 4 to 12 and m isindependently an integer from 4 to 12. In embodiments,-L^(9C)-L^(9D)-L^(9E)- is independently

wherein n is independently an integer from 4 to 12 and m isindependently an integer from 4 to 12.

In embodiments, L⁹ is independently

wherein L^(9A), L^(9C), L^(9D), and L^(9E) are as described herein,including in embodiments.

In embodiments, -L^(9A)-L^(9B)-L^(9C)- is independently

wherein n is independently an integer from 4 to 12. In embodiments,-L^(9A)-L^(9B)-L^(9C)- is independently

wherein n is independently an integer from 4 to 12. In embodiments,-L^(9A)-L^(9B)-L^(9C)- is independently

wherein n is independently an integer from 4 to 12. In embodiments,-L^(9A)-L^(9B)-L^(9C)- is independently

wherein n is independently an integer from 4 to 12. In embodiments,-L^(9A)-L^(9B)-L^(9C)- is independently

wherein n is independently an integer from 4 to 12. In embodiments,-L^(9A)-L^(9B)-L^(9C)- is independently

wherein n is independently an integer from 4 to 12. In embodiments,-L^(9A)-L^(9B)-L^(9C)- is independently

wherein n is independently an integer from 4 to 12. In embodiments,-L^(9A)-L^(9B)-L^(9C)- is independently

wherein n is independently an integer from 4 to 12. In embodiments,-L^(9A)-L^(9B)-L^(9C)- is independently

wherein n is independently an integer from 4 to 12. In embodiments,-L^(9A)-L^(9B)-L^(9C)- is independently

wherein n is independently an integer from 4 to 12. In embodiments,-L^(9A)-L^(9B)-L^(9C)- is independently

wherein n is independently an integer from 4 to 12. In embodiments,-L^(9A)-L^(9B)-L^(9C)- is independently

wherein n is independently an integer from 4 to 12. In embodiments,-L^(9A)-L^(9B)-L^(9C)- is independently

wherein n is independently an integer from 4 to 12. In embodiments,-L^(9A)-L^(9B)-L^(9C)- is independently

wherein n is independently an integer from 4 to 12. In embodiments,-L^(9A)-L^(9B)-L^(9C)- is independently

wherein n is independently an integer from 4 to 12. In embodiments,-L^(9A)-L^(9B)-L^(9C)- is independently

wherein n is independently an integer from 4 to 12.

In embodiments, L^(9D) is independently —S—S— and L^(9E) isindependently an

and unsubstituted C₄-C₈ alkylene. In embodiments, L^(9D) isindependently and L^(9E) is independently an unsubstituted C₄-C₈alkylene.

In embodiments, L⁹ is independently

In embodiments, L⁹ is independently

In embodiments, L⁹ is independently

In embodiments, L⁹ is independently

The symbol n is independently an integer from 4 to 12, and the symbol mis independently an integer from 4 to 12. In embodiments, each L⁹ is thesame.

In embodiments, R⁹ is independently 1) an oligonucleotide moiety; or 2)a second non-reactive moiety selected from hydrogen, halogen, —CX⁹ ₃,—CHX⁹ ₂, —CH₂X⁹, —OCX⁹ ₃, —OCH₂X⁹, —OCHX⁹ ₂, —CN, —SO₉R^(9D),SO_(v9)NR^(9A)R^(9B), —NHC(O)NR^(9A)R^(9B), —N(O)_(m9), —NR^(9A)R^(9B),—C(O)R^(9C), —C(O)—OR^(9C), —C(O)NR^(9A)R^(9B), —OR^(9D),—NR^(9A)SO₂R^(9D), —NR^(9A)C(O)R^(9C), —NR^(9A)C(O)OR^(9C),—NR^(9A)OR^(9C), —OC(O)R^(9C), substituted or unsubstituted alkyl (e.g.,C₁-C₈, C₁-C₆, C₁-C₄, or C₁-C₂), substituted or unsubstituted heteroalkyl(e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3membered, or 4 to 5 membered), substituted or unsubstituted cycloalkyl(e.g., C₃-C₈, C₃-C₆, C₄-C₆, or C₅-C₆), substituted or unsubstitutedheterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6membered, 4 to 5 membered, or 5 to 6 membered), substituted orunsubstituted aryl (e.g., C₆-C₁₀ or phenyl), or substituted orunsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to 9 membered, or 5to 6 membered). R^(9A), R^(9B), R^(9C), and R^(9D) are independentlyhydrogen, —CX₃, —CN, —COOH, —CONH₂, —CHX₂, —CH₂X, substituted orunsubstituted alkyl (e.g., C₁-C₈, C₁-C₆, C₁-C₄, or C₁-C₂), substitutedor unsubstituted heteroalkyl (e.g., 2 to 8 membered, 2 to 6 membered, 4to 6 membered, 2 to 3 membered, or 4 to 5 membered), substituted orunsubstituted cycloalkyl (e.g., C₃-C₈, C₃-C₆, C₄-C₆, or C₅-C₆),substituted or unsubstituted heterocycloalkyl (e.g., 3 to 8 membered, 3to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered),substituted or unsubstituted aryl (e.g., C₆-C₁₀ or phenyl), substitutedor unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to 9 membered, or5 to 6 membered), a protecting group, or a leaving group; R^(9A) andR^(9B) substituents bonded to the same nitrogen atom may optionally bejoined to form a substituted or unsubstituted heterocycloalkyl (e.g., 3to 8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5to 6 membered) or substituted or unsubstituted heteroaryl (e.g., 5 to 10membered, 5 to 9 membered, or 5 to 6 membered). X and X⁹ areindependently —F, —Cl, —Br, or —I. The symbol n9 is independently aninteger from 0 to 4. The symbols m9 and v9 are each independently aninteger from 1 to 2. In embodiments, R⁹ is independently anoligonucleotide moiety. In embodiments, when R²² and R⁹ are eachindependently an oligonucleotide moiety, the R²² and R⁹ oligonucleotidemoieties are the same. In embodiments, when R²² and R⁹ are eachindependently an oligonucleotide moiety, the R²² and R⁹ oligonucleotidemoieties are different. In embodiments, when R²⁴ is a first non-reactivemoiety and R⁹ is a second non-reactive moiety, R²⁴ and R⁹ are different.In embodiments, when R²⁴ is a first non-reactive moiety and R⁹ is asecond non-reactive moiety, R²⁴ and R⁹ are the same.

In embodiments, a substituted R⁹ (e.g., substituted alkyl, substitutedheteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl,substituted aryl, and/or substituted heteroaryl) is substituted with atleast one substituent group, size-limited substituent group, or lowersubstituent group; wherein if the substituted R⁹ is substituted with aplurality of groups selected from substituent groups, size-limitedsubstituent groups, and lower substituent groups; each substituentgroup, size-limited substituent group, and/or lower substituent groupmay optionally be different. In embodiments, when R⁹ is substituted, itis substituted with at least one substituent group. In embodiments, whenR⁹ is substituted, it is substituted with at least one size-limitedsubstituent group. In embodiments, when R⁹ is substituted, it issubstituted with at least one lower substituent group.

In embodiments, a substituted R^(9A) (e.g., substituted alkyl,substituted heteroalkyl, substituted cycloalkyl, substitutedheterocycloalkyl, substituted aryl, and/or substituted heteroaryl) issubstituted with at least one substituent group, size-limitedsubstituent group, or lower substituent group; wherein if thesubstituted R^(9A) is substituted with a plurality of groups selectedfrom substituent groups, size-limited substituent groups, and lowersubstituent groups; each substituent group, size-limited substituentgroup, and/or lower substituent group may optionally be different. Inembodiments, when R^(9A) is substituted, it is substituted with at leastone substituent group. In embodiments, when R^(9A) is substituted, it issubstituted with at least one size-limited substituent group. Inembodiments, when R^(9A) is substituted, it is substituted with at leastone lower substituent group.

In embodiments, a substituted R^(9B) (e.g., substituted alkyl,substituted heteroalkyl, substituted cycloalkyl, substitutedheterocycloalkyl, substituted aryl, and/or substituted heteroaryl) issubstituted with at least one substituent group, size-limitedsubstituent group, or lower substituent group; wherein if thesubstituted R^(9B) is substituted with a plurality of groups selectedfrom substituent groups, size-limited substituent groups, and lowersubstituent groups; each substituent group, size-limited substituentgroup, and/or lower substituent group may optionally be different. Inembodiments, when R^(9B) is substituted, it is substituted with at leastone substituent group. In embodiments, when R^(9B) is substituted, it issubstituted with at least one size-limited substituent group. Inembodiments, when R^(9B) is substituted, it is substituted with at leastone lower substituent group.

In embodiments, a substituted ring formed when R^(9A) and R^(9B)substituents bonded to the same nitrogen atom are joined (e.g.,substituted heterocycloalkyl and/or substituted heteroaryl) issubstituted with at least one substituent group, size-limitedsubstituent group, or lower substituent group; wherein if thesubstituted ring formed when R^(9A) and R^(9B) substituents bonded tothe same nitrogen atom are joined is substituted with a plurality ofgroups selected from substituent groups, size-limited substituentgroups, and lower substituent groups; each substituent group,size-limited substituent group, and/or lower substituent group mayoptionally be different. In embodiments, when the substituted ringformed when R^(9A) and R^(9B) substituents bonded to the same nitrogenatom are joined is substituted, it is substituted with at least onesubstituent group. In embodiments, when the substituted ring formed whenR^(9A) and R^(9B) substituents bonded to the same nitrogen atom arejoined is substituted, it is substituted with at least one size-limitedsubstituent group. In embodiments, when the substituted ring formed whenR^(9A) and R^(9B) substituents bonded to the same nitrogen atom arejoined is substituted, it is substituted with at least one lowersubstituent group.

In embodiments, a substituted R^(9C) (e.g., substituted alkyl,substituted heteroalkyl, substituted cycloalkyl, substitutedheterocycloalkyl, substituted aryl, and/or substituted heteroaryl) issubstituted with at least one substituent group, size-limitedsubstituent group, or lower substituent group; wherein if thesubstituted R^(9C) is substituted with a plurality of groups selectedfrom substituent groups, size-limited substituent groups, and lowersubstituent groups; each substituent group, size-limited substituentgroup, and/or lower substituent group may optionally be different. Inembodiments, when R^(9C) is substituted, it is substituted with at leastone substituent group. In embodiments, when R^(9C) is substituted, it issubstituted with at least one size-limited substituent group. Inembodiments, when R^(9C) is substituted, it is substituted with at leastone lower substituent group.

In embodiments, a substituted R^(9D) (e.g., substituted alkyl,substituted heteroalkyl, substituted cycloalkyl, substitutedheterocycloalkyl, substituted aryl, and/or substituted heteroaryl) issubstituted with at least one substituent group, size-limitedsubstituent group, or lower substituent group; wherein if thesubstituted R^(9D) is substituted with a plurality of groups selectedfrom substituent groups, size-limited substituent groups, and lowersubstituent groups; each substituent group, size-limited substituentgroup, and/or lower substituent group may optionally be different. Inembodiments, when R^(9D) is substituted, it is substituted with at leastone substituent group. In embodiments, when R^(9D) is substituted, it issubstituted with at least one size-limited substituent group. Inembodiments, when R^(9D) is substituted, it is substituted with at leastone lower substituent group.

In embodiments, R⁹ is independently a non-reactive moiety selected fromhalogen, —CX⁹ ₃, —CHX⁹ ₂, —CH₂X⁹, —OCX⁹ ₃, —OCH₂X⁹, —OCHX⁹ ₂, —CN,—SO_(n9)R^(9D)—SO_(v9)NR^(9A)R^(9B), —NHC(O)NR^(9A)R^(9B), —N(O)_(m9),—NR^(9A)R^(9B), —C(O)R^(9C), —C(O)—OR^(9C), —C(O)NR^(9A)R^(9B),—OR^(9D), —NR^(9A)SO₂R^(9D), —NR^(9A)C(O)R^(9C), —NR^(9A)C(O)OR^(9C),—NR^(9A)OR^(9C), —OC(O)R^(9C), substituted or unsubstituted alkyl (e.g.,C₁-C₈, C₁-C₆, C₁-C₄, or C₁-C₂), substituted or unsubstituted heteroalkyl(e.g., 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3membered, or 4 to 5 membered), substituted or unsubstituted cycloalkyl(e.g., C₃-C₈, C₃-C₆, C₄-C₆, or C₅-C₆), substituted or unsubstitutedheterocycloalkyl (e.g., 3 to 8 membered, 3 to 6 membered, 4 to 6membered, 4 to 5 membered, or 5 to 6 membered), substituted orunsubstituted aryl (e.g., C₆-C₁₀ or phenyl), or substituted orunsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to 9 membered, or 5to 6 membered). In embodiments, R⁹ is independently a non-reactivemoiety selected from halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CH₂Cl, —CH₂Br,—CH₂F, —CH₂I, —CHCl₂, —CHBr₂, —CHF₂, —CHI₂, —CN, —OH, —NH₂, —COOH,—CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂,—NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃, —OCBr₃, —OCF₃,—OCI₃, —OCH₂Cl, —OCH₂Br, —OCH₂F, —OCH₂I, —OCHCl₂, —OCHBr₂, —OCHF₂,—OCHI₂, substituted or unsubstituted alkyl (e.g., C₁-C₈, C₁-C₆, C₁-C₄,or C₁-C₂), substituted or unsubstituted heteroalkyl (e.g., 2 to 8membered, 2 to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4 to 5membered), substituted or unsubstituted cycloalkyl (e.g., C₃-C₈, C₃-C₆,C₄-C₆, or C₅-C₆), substituted or unsubstituted heterocycloalkyl (e.g., 3to 8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5to 6 membered), substituted or unsubstituted aryl (e.g., C₆-C₁₀ orphenyl), or substituted or unsubstituted heteroaryl (e.g., 5 to 10membered, 5 to 9 membered, or 5 to 6 membered). In embodiments, R⁹ isindependently hydrogen, —OCH₂CH₂—(OCH₂CH₂)_(p)—OCH₃ or —N(CH₃)₂; and pis independently an integer from 0 to 10. In embodiments, each R⁹ is thesame.

In embodiments, -L⁹-R⁹ is independently —OCH₂CH₂—(OCH₂CH₂)_(p)—OCH₃ or—N(CH₃)₂; and p is independently an integer from 4 to 10.

In embodiments, R¹⁰ is independently —CN. In embodiments, R¹⁰ isindependently substituted or unsubstituted alkyl. In embodiments, R¹⁰ isindependently substituted or unsubstituted C₁-C₄ alkyl. In embodiments,R¹⁰ is independently unsubstituted C₁-C₄ alkyl. In embodiments, R¹⁰ isindependently unsubstituted methyl. In embodiments, R¹⁰ is independentlyunsubstituted ethyl. In embodiments, R¹⁰ is independently unsubstitutedpropyl. In embodiments, R¹⁰ is independently unsubstituted n-propyl. Inembodiments, R¹⁰ is independently unsubstituted isopropyl. Inembodiments, R¹⁰ is independently unsubstituted butyl. In embodiments,R¹⁰ is independently unsubstituted n-butyl. In embodiments, R¹⁰ isindependently unsubstituted tert-butyl. In embodiments, R¹⁰ isindependently substituted or unsubstituted heteroalkyl. In embodiments,R¹⁰ is independently substituted or unsubstituted 2 to 6 memberedheteroalkyl. In embodiments, each R¹⁰ is the same.

In embodiments, L¹¹ is independently a substituted or unsubstitutedheteroalkylene. In embodiments, L^(11A) is independently a bond. Inembodiments, L^(11B) and L^(ID) are independently substituted orunsubstituted heteroalkylene; L^(11C) is independently a substituted orunsubstituted heteroarylene; and L^(11E) is independently a bond. Inembodiments, L^(11B) and L^(11E) are independently substituted orunsubstituted heteroalkylene; L^(11C) is independently a substituted orunsubstituted heteroarylene; and L^(ID) is independently a substitutedor unsubstituted arylene. In embodiments, L^(11A) is independently asubstituted or unsubstituted heteroalkylene.

In embodiments, L¹¹ is independently or

In embodiments, L¹² is independently a substituted or unsubstitutedheteroalkylene. In embodiments, L^(12B) and L^(12D) are independentlysubstituted or unsubstituted heteroalkylene; L^(12C) is independently asubstituted or unsubstituted heteroarylene; and L^(12E) is independentlya bond. In embodiments, L^(12B) and L^(12E) are independentlysubstituted or unsubstituted heteroalkylene; L^(12C) is independently asubstituted or unsubstituted heteroarylene; and L^(12D) is independentlya substituted or unsubstituted arylene. In embodiments, L^(12A) isindependently a bond.

In embodiments L¹² is independently

In embodiments, L¹² is independently

In embodiments, L¹² is independently

In embodiments, L¹² is independently

In embodiments, L¹² is independently

In embodiments, L¹² is independently

In embodiments, L¹² is independently

In embodiments, L¹² is independently

In embodiments, L¹² is independently

In embodiments, L¹² is independently

In embodiments, L¹² is independently

In embodiments, L¹² is independently

In embodiments, L¹² is independently

In embodiments, L¹² is independently

In embodiments, L¹² is independently

In embodiments, each L¹² is the same.

In embodiments, R¹² and R¹³ are independently hydrogen. In embodiments,R¹² is independently hydrogen. In embodiments, R¹² is independentlysubstituted or unsubstituted C₁-C₄ alkyl. In embodiments, R¹² isindependently unsubstituted C₁-C₄ alkyl. In embodiments, R¹² isindependently unsubstituted methyl. In embodiments, R¹² is independentlyunsubstituted ethyl. In embodiments, R¹² is independently unsubstitutedpropyl. In embodiments, R¹² is independently unsubstituted n-propyl. Inembodiments, R¹² is independently unsubstituted isopropyl. Inembodiments, R¹² is independently unsubstituted butyl. In embodiments,R¹² is independently unsubstituted n-butyl. In embodiments, R¹² isindependently unsubstituted tert-butyl. In embodiments, each R¹² is thesame. In embodiments, R¹³ is independently hydrogen. In embodiments, R¹³is independently substituted or unsubstituted C₁-C₄ alkyl. Inembodiments, R¹³ is independently unsubstituted C₁-C₄ alkyl. Inembodiments, R¹³ is independently unsubstituted methyl. In embodiments,R¹³ is independently unsubstituted ethyl. In embodiments, R¹³ isindependently unsubstituted propyl. In embodiments, R¹³ is independentlyunsubstituted n-propyl. In embodiments, R¹³ is independentlyunsubstituted isopropyl. In embodiments, R¹³ is independentlyunsubstituted butyl. In embodiments, R¹³ is independently unsubstitutedn-butyl. In embodiments, R¹³ is independently unsubstituted tert-butyl.In embodiments, each R¹³ is the same.

In embodiments, R¹⁴ is independently halogen. In embodiments, R¹⁴ isindependently —Br. In embodiments, R¹⁴ is independently —OH. Inembodiments, R¹⁴ is independently substituted or unsubstituted alkyl. Inembodiments, R¹⁴ is independently substituted or unsubstituted C₁-C₄alkyl. In embodiments, R¹⁴ is independently unsubstituted C₁-C₄ alkyl.In embodiments, R¹⁴ is independently unsubstituted methyl. Inembodiments, R¹⁴ is independently unsubstituted ethyl. In embodiments,R¹⁴ is independently unsubstituted propyl. In embodiments, R¹⁴ isindependently unsubstituted n-propyl. In embodiments, R¹⁴ isindependently unsubstituted isopropyl. In embodiments, R¹⁴ isindependently unsubstituted butyl. In embodiments, R¹⁴ is independentlyunsubstituted n-butyl. In embodiments, R¹⁴ is independentlyunsubstituted tert-butyl. In embodiments, R¹⁴ is independentlysubstituted or unsubstituted heteroalkyl. In embodiments, R¹⁴ isindependently substituted or unsubstituted 2 to 6 membered heteroalkyl.In embodiments, R¹⁴ is independently substituted or unsubstituted C₁-C₄alkyl. In embodiments, R¹⁴ is independently —O-(unsubstituted C₁-C₄alkyl). In embodiments, R¹⁴ is independently unsubstituted methoxy. Inembodiments, R¹⁴ is independently unsubstituted ethoxy. In embodiments,R¹⁴ is independently unsubstituted propoxy. In embodiments, R¹⁴ isindependently unsubstituted n-propoxy. In embodiments, R¹⁴ isindependently unsubstituted isopropoxy. In embodiments, R¹⁴ isindependently unsubstituted butoxy. In embodiments, R¹⁴ is independentlyunsubstituted n-butoxy. In embodiments, R¹⁴ is independentlyunsubstituted tert-butoxy. In embodiments, R¹⁴ is independently—C(CH₃)₂CN. In embodiments, R¹⁴ is independently —CH₂CN. In embodiments,R¹⁴ is independently —CH₂Ph. In embodiments, R¹⁴ is independently

In embodiments, R¹⁴ is independently

In embodiments, R¹⁴ is independently

In embodiments, R¹⁴ is independently

In embodiments, R¹⁴ is independently

In embodiments, R¹⁴ is independently

In embodiments, R¹⁴ is independently

In embodiments, each R¹⁴ is the same.

In embodiments, R¹⁵ is independently —CN. In embodiments, R¹⁵ isindependently substituted or unsubstituted alkyl. In embodiments, R¹⁵ isindependently substituted or unsubstituted C₁-C₄ alkyl. In embodiments,R¹⁵ is independently unsubstituted C₁-C₄ alkyl. In embodiments, R¹⁵ isindependently unsubstituted methyl. In embodiments, R¹⁵ is independentlyunsubstituted ethyl. In embodiments, R¹⁵ is independently unsubstitutedpropyl. In embodiments, R¹⁵ is independently unsubstituted n-propyl. Inembodiments, R¹⁵ is independently unsubstituted isopropyl. Inembodiments, R¹⁵ is independently unsubstituted butyl. In embodiments,R¹⁵ is independently unsubstituted n-butyl. In embodiments, R¹⁵ isindependently unsubstituted tert-butyl. In embodiments, R¹⁵ isindependently substituted or unsubstituted heteroalkyl. In embodiments,R¹⁵ is independently substituted or unsubstituted 2 to 6 memberedheteroalkyl. In embodiments, each R¹⁵ is the same.

In embodiments, L¹⁶ is independently substituted or unsubstitutedheteroalkylene. In embodiments, L^(16B) and L^(16D) are independentlysubstituted or unsubstituted heteroalkylene; L^(16C) is independently asubstituted or unsubstituted heteroarylene; and L^(16E) is independentlya bond. In embodiments, L^(16B) and L^(16E) are independentlysubstituted or unsubstituted heteroalkylene; L^(16C) is independently asubstituted or unsubstituted heteroarylene; and L^(16D) is independentlya substituted or unsubstituted arylene. In embodiments, L^(16A) isindependently a bond. In embodiments, each L¹⁶ is the same.

In embodiments, R¹⁶ is independently a non-reactive moiety selected fromhydrogen, halogen, —CCl₃, —CBr₃, —CF₃, —CI₃, —CH₂Cl, —CH₂Br, —CH₂F,—CH₂I, —CHCl₂, —CHBr₂, —CHF₂, —CHI₂, —CN, —OH, —NH₂, —COOH, —CONH₂,—NO₂, —SH, —SO₃H, —SO₄H, —SO₂ NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂,—NHC(O)NH₂, —NHSO₂H, —NHC(O)H, —NHC(O)OH, —NHOH, —OCCl₃, —OCBr₃, —OCF₃,—OCI₃, —OCH₂Cl, —OCH₂Br, —OCH₂F, —OCH₂I, —OCHCl₂, —OCHBr₂, —OCHF₂,—OCHI₂, substituted or unsubstituted alkyl (e.g., C₁-C₈, C₁-C₆, C₁-C₄,or C₁-C₂), substituted or unsubstituted heteroalkyl (e.g., 2 to 8membered, 2 to 6 membered, 4 to 6 membered, 2 to 3 membered, or 4 to 5membered), substituted or unsubstituted cycloalkyl (e.g., C₃-C₈, C₃-C₆,C₄-C₆, or C₅-C₆), substituted or unsubstituted heterocycloalkyl (e.g., 3to 8 membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5to 6 membered), substituted or unsubstituted aryl (e.g., C₆-C₁₀ orphenyl), or substituted or unsubstituted heteroaryl (e.g., 5 to 10membered, 5 to 9 membered, or 5 to 6 membered). In embodiments, R¹⁶ isindependently a non-reactive moiety selected from hydrogen, substitutedor unsubstituted alkyl (e.g., C₁-C₈, C₁-C₆, C₁-C₄, or C₁-C₂),substituted or unsubstituted heteroalkyl (e.g., 2 to 8 membered, 2 to 6membered, 4 to 6 membered, 2 to 3 membered, or 4 to 5 membered),substituted or unsubstituted cycloalkyl (e.g., C₃-C₈, C₃-C₆, C₄-C₆, orC₅-C₆), substituted or unsubstituted heterocycloalkyl (e.g., 3 to 8membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6membered), substituted or unsubstituted aryl (e.g., C₆-C₁₀ or phenyl),or substituted or unsubstituted heteroaryl (e.g., 5 to 10 membered, 5 to9 membered, or 5 to 6 membered). In embodiments, R¹⁶ is independentlyhydrogen, —OCH₂CH₂—(OCH₂CH₂)_(p)—OCH₃ or —N(CH₃)₂; and p is an integerfrom 0 to 10. In embodiments, p is an integer from 1 to 10. Inembodiments, each R¹⁶ is the same.

In embodiments, when R²⁴ is a first non-reactive moiety, R⁹ is a secondnon-reactive moiety, and R¹⁶ is a third non-reactive moiety, R²⁴, R⁹,and R¹⁶ are different. In embodiments, when R²⁴ is a first non-reactivemoiety, R⁹ is a second non-reactive moiety, and R¹⁶ is a thirdnon-reactive moiety, R²⁴, R⁹, and R¹⁶ are the same. In embodiments, whenR²⁴ is a non-reactive moiety and R¹⁶ is a non-reactive moiety, R²⁴ andR¹⁶ are different. In embodiments, when R²⁴ is a non-reactive moiety andR¹⁶ is a non-reactive moiety, R²⁴ and R¹⁶ are the same.

In embodiments, -L¹⁶-R¹⁶ is independently —OCH₂CH₂—(OCH₂CH₂)_(p)—OCH₃ or—N(CH₃)₂; and p is an integer from 4 to 10. In embodiments, R¹⁷ and R¹⁸are independently hydrogen.

In embodiments, R¹⁷ is independently hydrogen. In embodiments, R¹⁷ isindependently substituted or unsubstituted C₁-C₄ alkyl. In embodiments,R¹⁷ is independently unsubstituted C₁-C₄ alkyl. In embodiments, R¹⁷ isindependently unsubstituted methyl. In embodiments, R¹⁷ is independentlyunsubstituted ethyl. In embodiments, R¹⁷ is independently unsubstitutedpropyl. In embodiments, R¹⁷ is independently unsubstituted n-propyl. Inembodiments, R¹⁷ is independently unsubstituted isopropyl. Inembodiments, R¹⁷ is independently unsubstituted butyl. In embodiments,R¹⁷ is independently unsubstituted n-butyl. In embodiments, R¹⁷ isindependently unsubstituted tert-butyl. In embodiments, each R¹⁷ is thesame. In embodiments, R¹⁸ is independently hydrogen. In embodiments, R¹⁸is independently substituted or unsubstituted C₁-C₄ alkyl. Inembodiments, R¹⁸ is independently unsubstituted C₁-C₄ alkyl. Inembodiments, R¹⁸ is independently unsubstituted methyl. In embodiments,R¹⁸ is independently unsubstituted ethyl. In embodiments, R¹⁸ isindependently unsubstituted propyl. In embodiments, R¹⁸ is independentlyunsubstituted n-propyl. In embodiments, R¹⁸ is independentlyunsubstituted isopropyl. In embodiments, R¹⁸ is independentlyunsubstituted butyl. In embodiments, R¹⁸ is independently unsubstitutedn-butyl. In embodiments, R¹⁸ is independently unsubstituted tert-butyl.In embodiments, each R¹⁸ is the same. In embodiments, R¹⁹ isindependently halogen. In embodiments, R¹⁹ is independently —Br. Inembodiments, R¹⁹ is independently —OH. In embodiments, R¹⁹ isindependently substituted or unsubstituted alkyl. In embodiments, R¹⁹ isindependently substituted or unsubstituted C₁-C₄ alkyl. In embodiments,R¹⁹ is independently unsubstituted C₁-C₄ alkyl. In embodiments, R¹⁹ isindependently unsubstituted methyl. In embodiments, R¹⁹ is independentlyunsubstituted ethyl. In embodiments, R¹⁹ is independently unsubstitutedpropyl. In embodiments, R¹⁹ is independently unsubstituted n-propyl. Inembodiments, R¹⁹ is independently unsubstituted isopropyl. Inembodiments, R¹⁹ is independently unsubstituted butyl. In embodiments,R¹⁹ is independently unsubstituted n-butyl. In embodiments, R¹⁹ isindependently unsubstituted tert-butyl. In embodiments, R¹⁹ isindependently substituted or unsubstituted heteroalkyl. In embodiments,R¹⁹ is independently substituted or unsubstituted 2 to 6 memberedheteroalkyl. In embodiments, R¹⁹ is independently substituted orunsubstituted C₁-C₄ alkyl. In embodiments, R¹⁹ is independently—O-(unsubstituted C₁-C₄ alkyl). In embodiments, R¹⁹ is independentlyunsubstituted methoxy. In embodiments, R¹⁹ is independentlyunsubstituted ethoxy. In embodiments, R¹⁹ is independently unsubstitutedpropoxy. In embodiments, R¹⁹ is independently unsubstituted n-propoxy.In embodiments, R¹⁹ is independently unsubstituted isopropoxy. Inembodiments, R¹⁹ is independently unsubstituted butoxy. In embodiments,R¹⁹ is independently unsubstituted n-butoxy. In embodiments, R¹⁹ isindependently unsubstituted tert-butoxy. In embodiments, R¹⁹ isindependently —C(CH₃)₂CN. In embodiments, R¹⁹ is independently —CH₂CN.In embodiments, R¹⁹ is independently —CH₂Ph. In embodiments, R¹⁹ isindependently

In embodiments, R¹⁹ is independently

In embodiments, R¹⁹ is independently

In embodiments, R¹⁹ is independently

In embodiments, R¹⁹ is independently

In embodiments, R¹⁹ is independently

In embodiments, R¹⁹ is independently

In embodiments, each R¹⁹ is the same.

In embodiments, z1a is independently an integer from 0 to 10. Inembodiments, z1a is independently an integer from 0 to 20. Inembodiments, z1a is independently an integer from 0 to 30. Inembodiments, z1a is independently an integer from 0 to 40. Inembodiments, z1a is independently an integer from 0 to 50. Inembodiments, z1a is independently an integer from 0 to 60. Inembodiments, z1a is independently an integer from 0 to 70. Inembodiments, z1a is independently an integer from 0 to 80. Inembodiments, z1a is independently an integer from 0 to 90. Inembodiments, z1a is independently an integer from 0 to 100. Inembodiments, z1a is independently an integer from 0 to 150. Inembodiments, z1a is independently an integer from 0 to 200. Inembodiments, z1a is independently an integer from 0 to 250. Inembodiments, z1a is independently an integer from 0 to 300. Inembodiments, z1a is independently an integer from 0 to 350. Inembodiments, z1a is independently an integer from 0 to 400. Inembodiments, z1a is independently an integer from 0 to 450. Inembodiments, z1a is independently an integer from 0 to 500. Inembodiments, z1a is independently an integer from 0 to 550. Inembodiments, z1a is independently an integer from 0 to 600. Inembodiments, z1a is independently an integer from 0 to 650. Inembodiments, z1a is independently an integer from 0 to 700. Inembodiments, z1a is independently an integer from 0 to 750. Inembodiments, z1a is independently an integer from 0 to 800. Inembodiments, z1a is independently an integer from 0 to 850. Inembodiments, z1a is independently an integer from 0 to 900. Inembodiments, z1a is independently an integer from 0 to 950. Inembodiments, z1a is independently an integer from 0 to 1000. Inembodiments, z1a is independently an integer from 0 to 1500. Inembodiments, z1a is independently an integer from 0 to 2000. Inembodiments, z1a is independently an integer from 0 to 2500. Inembodiments, z1a is independently an integer from 0 to 3000. Inembodiments, z1a is independently an integer from 0 to 3500. Inembodiments, z1a is independently an integer from 0 to 4000. Inembodiments, z1a is independently an integer from 0 to 4500. Inembodiments, z1a is independently an integer from 0 to 5000. Inembodiments, z1a is independently an integer from 1 to 10. Inembodiments, z1a is independently an integer from 1 to 20. Inembodiments, z1a is independently an integer from 1 to 30. Inembodiments, z1a is independently an integer from 1 to 40. Inembodiments, z1a is independently an integer from 1 to 50. Inembodiments, z1a is independently an integer from 1 to 60. Inembodiments, z1a is independently an integer from 1 to 70. Inembodiments, z1a is independently an integer from 1 to 80. Inembodiments, z1a is independently an integer from 1 to 90. Inembodiments, z1a is independently an integer from 1 to 100. Inembodiments, z1a is independently an integer from 1 to 150. Inembodiments, z1a is independently an integer from 1 to 200. Inembodiments, z1a is independently an integer from 1 to 250. Inembodiments, z1a is independently an integer from 1 to 300. Inembodiments, z1a is independently an integer from 1 to 350. Inembodiments, z1a is independently an integer from 1 to 400. Inembodiments, z1a is independently an integer from 1 to 450. Inembodiments, z1a is independently an integer from 1 to 500. Inembodiments, z1a is independently an integer from 1 to 550. Inembodiments, z1a is independently an integer from 1 to 600. Inembodiments, z1a is independently an integer from 1 to 650. Inembodiments, z1a is independently an integer from 1 to 700. Inembodiments, z1a is independently an integer from 1 to 750. Inembodiments, z1a is independently an integer from 1 to 800. Inembodiments, z1a is independently an integer from 1 to 850. Inembodiments, z1a is independently an integer from 1 to 900. Inembodiments, z1a is independently an integer from 1 to 950. Inembodiments, z1a is independently an integer from 1 to 1000. Inembodiments, z1a is independently an integer from 1 to 1500. Inembodiments, z1a is independently an integer from 1 to 2000. Inembodiments, z1a is independently an integer from 1 to 2500. Inembodiments, z1a is independently an integer from 1 to 3000. Inembodiments, z1a is independently an integer from 1 to 3500. Inembodiments, z1a is independently an integer from 1 to 4000. Inembodiments, z1a is independently an integer from 1 to 4500. Inembodiments, z1a is independently an integer from 1 to 5000. Inembodiments, z1b is independently an integer from 0 to 10. Inembodiments, z1b is independently an integer from 0 to 20. Inembodiments, z1b is independently an integer from 0 to 30. Inembodiments, z1b is independently an integer from 0 to 40. Inembodiments, z1b is independently an integer from 0 to 50. Inembodiments, z1b is independently an integer from 0 to 60. Inembodiments, z1b is independently an integer from 0 to 70. Inembodiments, z1b is independently an integer from 0 to 80. Inembodiments, z1b is independently an integer from 0 to 90. Inembodiments, z1b is independently an integer from 0 to 100. Inembodiments, z1b is independently an integer from 0 to 150. Inembodiments, z1b is independently an integer from 0 to 200. Inembodiments, z1b is independently an integer from 0 to 250. Inembodiments, z1b is independently an integer from 0 to 300. Inembodiments, z1b is independently an integer from 0 to 350. Inembodiments, z1b is independently an integer from 0 to 400. Inembodiments, z1b is independently an integer from 0 to 450. Inembodiments, z1b is independently an integer from 0 to 500. Inembodiments, z1b is independently an integer from 0 to 550. Inembodiments, z1b is independently an integer from 0 to 600. Inembodiments, z1b is independently an integer from 0 to 650. Inembodiments, z1b is independently an integer from 0 to 700. Inembodiments, z1b is independently an integer from 0 to 750. Inembodiments, z1b is independently an integer from 0 to 800. Inembodiments, z1b is independently an integer from 0 to 850. Inembodiments, z1b is independently an integer from 0 to 900. Inembodiments, z1b is independently an integer from 0 to 950. Inembodiments, z1b is independently an integer from 0 to 1000. Inembodiments, z1b is independently an integer from 0 to 1500. Inembodiments, z1b is independently an integer from 0 to 2000. Inembodiments, z1b is independently an integer from 0 to 2500. Inembodiments, z1b is independently an integer from 0 to 3000. Inembodiments, z1b is independently an integer from 0 to 3500. Inembodiments, z1b is independently an integer from 0 to 4000. Inembodiments, z1b is independently an integer from 0 to 4500. Inembodiments, z1b is independently an integer from 0 to 5000. Inembodiments, z1b is independently an integer from 1 to 10. Inembodiments, z1b is independently an integer from 1 to 20. Inembodiments, z1b is independently an integer from 1 to 30. Inembodiments, z1b is independently an integer from 1 to 40. Inembodiments, z1b is independently an integer from 1 to 50. Inembodiments, z1b is independently an integer from 1 to 60. Inembodiments, z1b is independently an integer from 1 to 70. Inembodiments, z1b is independently an integer from 1 to 80. Inembodiments, z1b is independently an integer from 1 to 90. Inembodiments, z1b is independently an integer from 1 to 100. Inembodiments, z1b is independently an integer from 1 to 150. Inembodiments, z1b is independently an integer from 1 to 200. Inembodiments, z1b is independently an integer from 1 to 250. Inembodiments, z1b is independently an integer from 1 to 300. Inembodiments, z1b is independently an integer from 1 to 350. Inembodiments, z1b is independently an integer from 1 to 400. Inembodiments, z1b is independently an integer from 1 to 450. Inembodiments, z1b is independently an integer from 1 to 500. Inembodiments, z1b is independently an integer from 1 to 550. Inembodiments, z1b is independently an integer from 1 to 600. Inembodiments, z1b is independently an integer from 1 to 650. Inembodiments, z1b is independently an integer from 1 to 700. Inembodiments, z1b is independently an integer from 1 to 750. Inembodiments, z1b is independently an integer from 1 to 800. Inembodiments, z1b is independently an integer from 1 to 850. Inembodiments, z1b is independently an integer from 1 to 900. Inembodiments, z1b is independently an integer from 1 to 950. Inembodiments, z1b is independently an integer from 1 to 1000. Inembodiments, z1b is independently an integer from 1 to 1500. Inembodiments, z1b is independently an integer from 1 to 2000. Inembodiments, z1b is independently an integer from 1 to 2500. Inembodiments, z1b is independently an integer from 1 to 3000. Inembodiments, z1b is independently an integer from 1 to 3500. Inembodiments, z1b is independently an integer from 1 to 4000. Inembodiments, z1b is independently an integer from 1 to 4500. Inembodiments, z1b is independently an integer from 1 to 5000. Inembodiments, z1c is independently an integer from 0 to 10. Inembodiments, z1c is independently an integer from 0 to 20. Inembodiments, z1c is independently an integer from 0 to 30. Inembodiments, z1c is independently an integer from 0 to 40. Inembodiments, z1c is independently an integer from 0 to 50. Inembodiments, z1c is independently an integer from 0 to 60. Inembodiments, z1c is independently an integer from 0 to 70. Inembodiments, z1c is independently an integer from 0 to 80. Inembodiments, z1c is independently an integer from 0 to 90. Inembodiments, z1c is independently an integer from 0 to 100. Inembodiments, z1c is independently an integer from 0 to 150. Inembodiments, z1c is independently an integer from 0 to 200. Inembodiments, z1c is independently an integer from 0 to 250. Inembodiments, z1c is independently an integer from 0 to 300. Inembodiments, z1c is independently an integer from 0 to 350. Inembodiments, z1c is independently an integer from 0 to 400. Inembodiments, z1c is independently an integer from 0 to 450. Inembodiments, z1c is independently an integer from 0 to 500. Inembodiments, z1c is independently an integer from 0 to 550. Inembodiments, z1c is independently an integer from 0 to 600. Inembodiments, z1c is independently an integer from 0 to 650. Inembodiments, z1c is independently an integer from 0 to 700. Inembodiments, z1c is independently an integer from 0 to 750. Inembodiments, z1c is independently an integer from 0 to 800. Inembodiments, z1c is independently an integer from 0 to 850. Inembodiments, z1c is independently an integer from 0 to 900. Inembodiments, z1c is independently an integer from 0 to 950. Inembodiments, z1c is independently an integer from 0 to 1000. Inembodiments, z1c is independently an integer from 0 to 1500. Inembodiments, z1c is independently an integer from 0 to 2000. Inembodiments, z1c is independently an integer from 0 to 2500. Inembodiments, z1c is independently an integer from 0 to 3000. Inembodiments, z1c is independently an integer from 0 to 3500. Inembodiments, z1c is independently an integer from 0 to 4000. Inembodiments, z1c is independently an integer from 0 to 4500. Inembodiments, z1c is independently an integer from 0 to 5000. Inembodiments, z1c is independently an integer from 1 to 10. Inembodiments, z1c is independently an integer from 1 to 20. Inembodiments, z1c is independently an integer from 1 to 30. Inembodiments, z1c is independently an integer from 1 to 40. Inembodiments, z1c is independently an integer from 1 to 50. Inembodiments, z1c is independently an integer from 1 to 60. Inembodiments, z1c is independently an integer from 1 to 70. Inembodiments, z1c is independently an integer from 1 to 80. Inembodiments, z1c is independently an integer from 1 to 90. Inembodiments, z1c is independently an integer from 1 to 100. Inembodiments, z1c is independently an integer from 1 to 150. Inembodiments, z1c is independently an integer from 1 to 200. Inembodiments, z1c is independently an integer from 1 to 250. Inembodiments, z1c is independently an integer from 1 to 300. Inembodiments, z1c is independently an integer from 1 to 350. Inembodiments, z1c is independently an integer from 1 to 400. Inembodiments, z1c is independently an integer from 1 to 450. Inembodiments, z1c is independently an integer from 1 to 500. Inembodiments, z1c is independently an integer from 1 to 550. Inembodiments, z1c is independently an integer from 1 to 600. Inembodiments, z1c is independently an integer from 1 to 650. Inembodiments, z1c is independently an integer from 1 to 700. Inembodiments, z1c is independently an integer from 1 to 750. Inembodiments, z1c is independently an integer from 1 to 800. Inembodiments, z1c is independently an integer from 1 to 850. Inembodiments, z1c is independently an integer from 1 to 900. Inembodiments, z1c is independently an integer from 1 to 950. Inembodiments, z1c is independently an integer from 1 to 1000. Inembodiments, z1c is independently an integer from 1 to 1500. Inembodiments, z1c is independently an integer from 1 to 2000. Inembodiments, z1c is independently an integer from 1 to 2500. Inembodiments, z1c is independently an integer from 1 to 3000. Inembodiments, z1c is independently an integer from 1 to 3500. Inembodiments, z1c is independently an integer from 1 to 4000. Inembodiments, z1c is independently an integer from 1 to 4500. Inembodiments, z1c is independently an integer from 1 to 5000. Inembodiments, z1d is independently an integer from 0 to 10. Inembodiments, z1d is independently an integer from 0 to 20. Inembodiments, z1d is independently an integer from 0 to 30. Inembodiments, z1d is independently an integer from 0 to 40. Inembodiments, z1d is independently an integer from 0 to 50. Inembodiments, z d is independently an integer from 0 to 60. Inembodiments, z d is independently an integer from 0 to 70. Inembodiments, z1d is independently an integer from 0 to 80. Inembodiments, z1d is independently an integer from 0 to 90. Inembodiments, z1d is independently an integer from 0 to 100. Inembodiments, z d is independently an integer from 0 to 150. Inembodiments, z1d is independently an integer from 0 to 200. Inembodiments, z1d is independently an integer from 0 to 250. Inembodiments, z1d is independently an integer from 0 to 300. Inembodiments, z1d is independently an integer from 0 to 350. Inembodiments, z1d is independently an integer from 0 to 400. Inembodiments, z1d is independently an integer from 0 to 450. Inembodiments, z1d is independently an integer from 0 to 500. Inembodiments, z1d is independently an integer from 0 to 550. Inembodiments, z1d is independently an integer from 0 to 600. Inembodiments, z1d is independently an integer from 0 to 650. Inembodiments, z1d is independently an integer from 0 to 700. Inembodiments, z1d is independently an integer from 0 to 750. Inembodiments, z1d is independently an integer from 0 to 800. Inembodiments, z1d is independently an integer from 0 to 850. Inembodiments, z1d is independently an integer from 0 to 900. Inembodiments, z1d is independently an integer from 0 to 950. Inembodiments, z1d is independently an integer from 0 to 1000. Inembodiments, z1d is independently an integer from 0 to 1500. Inembodiments, z1d is independently an integer from 0 to 2000. Inembodiments, z1d is independently an integer from 0 to 2500. Inembodiments, z1d is independently an integer from 0 to 3000. Inembodiments, z1d is independently an integer from 0 to 3500. Inembodiments, z1d is independently an integer from 0 to 4000. Inembodiments, z1d is independently an integer from 0 to 4500. Inembodiments, z1d is independently an integer from 0 to 5000. Inembodiments, z1d is independently an integer from 1 to 10. Inembodiments, z1d is independently an integer from 1 to 20. Inembodiments, z1d is independently an integer from 1 to 30. Inembodiments, z1d is independently an integer from 1 to 40. Inembodiments, z1d is independently an integer from 1 to 50. Inembodiments, z1d is independently an integer from 1 to 60. Inembodiments, z1d is independently an integer from 1 to 70. Inembodiments, z1d is independently an integer from 1 to 80. Inembodiments, z1d is independently an integer from 1 to 90. Inembodiments, z1d is independently an integer from 1 to 100. Inembodiments, z1d is independently an integer from 1 to 150. Inembodiments, z1d is independently an integer from 1 to 200. Inembodiments, z1d is independently an integer from 1 to 250. Inembodiments, z1d is independently an integer from 1 to 300. Inembodiments, z1d is independently an integer from 1 to 350. Inembodiments, z1d is independently an integer from 1 to 400. Inembodiments, z1d is independently an integer from 1 to 450. Inembodiments, z1d is independently an integer from 1 to 500. Inembodiments, z1d is independently an integer from 1 to 550. Inembodiments, z1d is independently an integer from 1 to 600. Inembodiments, z1d is independently an integer from 1 to 650. Inembodiments, z1d is independently an integer from 1 to 700. Inembodiments, z1d is independently an integer from 1 to 750. Inembodiments, z1d is independently an integer from 1 to 800. Inembodiments, z1d is independently an integer from 1 to 850. Inembodiments, z1d is independently an integer from 1 to 900. Inembodiments, z1d is independently an integer from 1 to 950. Inembodiments, z1d is independently an integer from 1 to 1000. Inembodiments, z1d is independently an integer from 1 to 1500. Inembodiments, z1d is independently an integer from 1 to 2000. Inembodiments, z1d is independently an integer from 1 to 2500. Inembodiments, z1d is independently an integer from 1 to 3000. Inembodiments, z1d is independently an integer from 1 to 3500. Inembodiments, z1d is independently an integer from 1 to 4000. Inembodiments, z1d is independently an integer from 1 to 4500. Inembodiments, z1d is independently an integer from 1 to 5000. Inembodiments, z2 is independently an integer from 0 to 10. Inembodiments, z2 is independently an integer from 0 to 20. Inembodiments, z2 is independently an integer from 0 to 30. Inembodiments, z2 is independently an integer from 0 to 40. Inembodiments, z2 is independently an integer from 0 to 50. Inembodiments, z2 is independently an integer from 0 to 60. Inembodiments, z2 is independently an integer from 0 to 70. Inembodiments, z2 is independently an integer from 0 to 80. Inembodiments, z2 is independently an integer from 0 to 90. Inembodiments, z2 is independently an integer from 0 to 100. Inembodiments, z2 is independently an integer from 0 to 150. Inembodiments, z2 is independently an integer from 0 to 200. Inembodiments, z2 is independently an integer from 0 to 250. Inembodiments, z2 is independently an integer from 0 to 300. Inembodiments, z2 is independently an integer from 0 to 350. Inembodiments, z2 is independently an integer from 0 to 400. Inembodiments, z2 is independently an integer from 0 to 450. Inembodiments, z2 is independently an integer from 0 to 500. Inembodiments, z2 is independently an integer from 0 to 550. Inembodiments, z2 is independently an integer from 0 to 600. Inembodiments, z2 is independently an integer from 0 to 650. Inembodiments, z2 is independently an integer from 0 to 700. Inembodiments, z2 is independently an integer from 0 to 750. Inembodiments, z2 is independently an integer from 0 to 800. Inembodiments, z2 is independently an integer from 0 to 850. Inembodiments, z2 is independently an integer from 0 to 900. Inembodiments, z2 is independently an integer from 0 to 950. Inembodiments, z2 is independently an integer from 0 to 1000. Inembodiments, z2 is independently an integer from 0 to 1500. Inembodiments, z2 is independently an integer from 0 to 2000. Inembodiments, z2 is independently an integer from 0 to 2500. Inembodiments, z2 is independently an integer from 0 to 3000. Inembodiments, z2 is independently an integer from 0 to 3500. Inembodiments, z2 is independently an integer from 0 to 4000. Inembodiments, z2 is independently an integer from 0 to 4500. Inembodiments, z2 is independently an integer from 0 to 5000. Inembodiments, z2 is independently an integer from 1 to 10. Inembodiments, z2 is independently an integer from 1 to 20. Inembodiments, z2 is independently an integer from 1 to 30. Inembodiments, z2 is independently an integer from 1 to 40. Inembodiments, z2 is independently an integer from 1 to 50. Inembodiments, z2 is independently an integer from 1 to 60. Inembodiments, z2 is independently an integer from 1 to 70. Inembodiments, z2 is independently an integer from 1 to 80. Inembodiments, z2 is independently an integer from 1 to 90. Inembodiments, z2 is independently an integer from 1 to 100. Inembodiments, z2 is independently an integer from 1 to 150. Inembodiments, z2 is independently an integer from 1 to 200. Inembodiments, z2 is independently an integer from 1 to 250. Inembodiments, z2 is independently an integer from 1 to 300. Inembodiments, z2 is independently an integer from 1 to 350. Inembodiments, z2 is independently an integer from 1 to 400. Inembodiments, z2 is independently an integer from 1 to 450. Inembodiments, z2 is independently an integer from 1 to 500. Inembodiments, z2 is independently an integer from 1 to 550. Inembodiments, z2 is independently an integer from 1 to 600. Inembodiments, z2 is independently an integer from 1 to 650. Inembodiments, z2 is independently an integer from 1 to 700. Inembodiments, z2 is independently an integer from 1 to 750. Inembodiments, z2 is independently an integer from 1 to 800. Inembodiments, z2 is independently an integer from 1 to 850. Inembodiments, z2 is independently an integer from 1 to 900. Inembodiments, z2 is independently an integer from 1 to 950. Inembodiments, z2 is independently an integer from 1 to 1000. Inembodiments, z2 is independently an integer from 1 to 1500. Inembodiments, z2 is independently an integer from 1 to 2000. Inembodiments, z2 is independently an integer from 1 to 2500. Inembodiments, z2 is independently an integer from 1 to 3000. Inembodiments, z2 is independently an integer from 1 to 3500. Inembodiments, z2 is independently an integer from 1 to 4000. Inembodiments, z2 is independently an integer from 1 to 4500. Inembodiments, z2 is independently an integer from 1 to 5000. Inembodiments, z3 is independently an integer from 0 to 10. Inembodiments, z3 is independently an integer from 0 to 20. Inembodiments, z3 is independently an integer from 0 to 30. Inembodiments, z3 is independently an integer from 0 to 40. Inembodiments, z3 is independently an integer from 0 to 50. Inembodiments, z3 is independently an integer from 0 to 60. Inembodiments, z3 is independently an integer from 0 to 70. Inembodiments, z3 is independently an integer from 0 to 80. Inembodiments, z3 is independently an integer from 0 to 90. Inembodiments, z3 is independently an integer from 0 to 100. Inembodiments, z3 is independently an integer from 0 to 150. Inembodiments, z3 is independently an integer from 0 to 200. Inembodiments, z3 is independently an integer from 0 to 250. Inembodiments, z3 is independently an integer from 0 to 300. Inembodiments, z3 is independently an integer from 0 to 350. Inembodiments, z3 is independently an integer from 0 to 400. Inembodiments, z3 is independently an integer from 0 to 450. Inembodiments, z3 is independently an integer from 0 to 500. Inembodiments, z3 is independently an integer from 0 to 550. Inembodiments, z3 is independently an integer from 0 to 600. Inembodiments, z3 is independently an integer from 0 to 650. Inembodiments, z3 is independently an integer from 0 to 700. Inembodiments, z3 is independently an integer from 0 to 750. Inembodiments, z3 is independently an integer from 0 to 800. Inembodiments, z3 is independently an integer from 0 to 850. Inembodiments, z3 is independently an integer from 0 to 900. Inembodiments, z3 is independently an integer from 0 to 950. Inembodiments, z3 is independently an integer from 0 to 1000. Inembodiments, z3 is independently an integer from 0 to 1500. Inembodiments, z3 is independently an integer from 0 to 2000. Inembodiments, z3 is independently an integer from 0 to 2500. Inembodiments, z3 is independently an integer from 0 to 3000. Inembodiments, z3 is independently an integer from 0 to 3500. Inembodiments, z3 is independently an integer from 0 to 4000. Inembodiments, z3 is independently an integer from 0 to 4500. Inembodiments, z3 is independently an integer from 0 to 5000. Inembodiments, z3 is independently an integer from 1 to 10. Inembodiments, z3 is independently an integer from 1 to 20. Inembodiments, z3 is independently an integer from 1 to 30. Inembodiments, z3 is independently an integer from 1 to 40. Inembodiments, z3 is independently an integer from 1 to 50. Inembodiments, z3 is independently an integer from 1 to 60. Inembodiments, z3 is independently an integer from 1 to 70. Inembodiments, z3 is independently an integer from 1 to 80. Inembodiments, z3 is independently an integer from 1 to 90. Inembodiments, z3 is independently an integer from 1 to 100. Inembodiments, z3 is independently an integer from 1 to 150. Inembodiments, z3 is independently an integer from 1 to 200. Inembodiments, z3 is independently an integer from 1 to 250. Inembodiments, z3 is independently an integer from 1 to 300. Inembodiments, z3 is independently an integer from 1 to 350. Inembodiments, z3 is independently an integer from 1 to 400. Inembodiments, z3 is independently an integer from 1 to 450. Inembodiments, z3 is independently an integer from 1 to 500. Inembodiments, z3 is independently an integer from 1 to 550. Inembodiments, z3 is independently an integer from 1 to 600. Inembodiments, z3 is independently an integer from 1 to 650. Inembodiments, z3 is independently an integer from 1 to 700. Inembodiments, z3 is independently an integer from 1 to 750. Inembodiments, z3 is independently an integer from 1 to 800. Inembodiments, z3 is independently an integer from 1 to 850. Inembodiments, z3 is independently an integer from 1 to 900. Inembodiments, z3 is independently an integer from 1 to 950. Inembodiments, z3 is independently an integer from 1 to 1000. Inembodiments, z3 is independently an integer from 1 to 1500. Inembodiments, z3 is independently an integer from 1 to 2000. Inembodiments, z3 is independently an integer from 1 to 2500. Inembodiments, z3 is independently an integer from 1 to 3000. Inembodiments, z3 is independently an integer from 1 to 3500. Inembodiments, z3 is independently an integer from 1 to 4000. Inembodiments, z3 is independently an integer from 1 to 4500. Inembodiments, z3 is independently an integer from 1 to 5000. Inembodiments, z4 is independently an integer from 0 to 10. Inembodiments, z4 is independently an integer from 0 to 20. Inembodiments, z4 is independently an integer from 0 to 30. Inembodiments, z4 is independently an integer from 0 to 40. Inembodiments, z4 is independently an integer from 0 to 50. Inembodiments, z4 is independently an integer from 0 to 60. Inembodiments, z4 is independently an integer from 0 to 70. Inembodiments, z4 is independently an integer from 0 to 80. Inembodiments, z4 is independently an integer from 0 to 90. Inembodiments, z4 is independently an integer from 0 to 100. Inembodiments, z4 is independently an integer from 0 to 150. Inembodiments, z4 is independently an integer from 0 to 200. Inembodiments, z4 is independently an integer from 0 to 250. Inembodiments, z4 is independently an integer from 0 to 300. Inembodiments, z4 is independently an integer from 0 to 350. Inembodiments, z4 is independently an integer from 0 to 400. Inembodiments, z4 is independently an integer from 0 to 450. Inembodiments, z4 is independently an integer from 0 to 500. Inembodiments, z4 is independently an integer from 0 to 550. Inembodiments, z4 is independently an integer from 0 to 600. Inembodiments, z4 is independently an integer from 0 to 650. Inembodiments, z4 is independently an integer from 0 to 700. Inembodiments, z4 is independently an integer from 0 to 750. Inembodiments, z4 is independently an integer from 0 to 800. Inembodiments, z4 is independently an integer from 0 to 850. Inembodiments, z4 is independently an integer from 0 to 900. Inembodiments, z4 is independently an integer from 0 to 950. Inembodiments, z4 is independently an integer from 0 to 1000. Inembodiments, z4 is independently an integer from 0 to 1500. Inembodiments, z4 is independently an integer from 0 to 2000. Inembodiments, z4 is independently an integer from 0 to 2500. Inembodiments, z4 is independently an integer from 0 to 3000. Inembodiments, z4 is independently an integer from 0 to 3500. Inembodiments, z4 is independently an integer from 0 to 4000. Inembodiments, z4 is independently an integer from 0 to 4500. Inembodiments, z4 is independently an integer from 0 to 5000. Inembodiments, z4 is independently an integer from 1 to 10. Inembodiments, z4 is independently an integer from 1 to 20. Inembodiments, z4 is independently an integer from 1 to 30. Inembodiments, z4 is independently an integer from 1 to 40. Inembodiments, z4 is independently an integer from 1 to 50. Inembodiments, z4 is independently an integer from 1 to 60. Inembodiments, z4 is independently an integer from 1 to 70. Inembodiments, z4 is independently an integer from 1 to 80. Inembodiments, z4 is independently an integer from 1 to 90. Inembodiments, z4 is independently an integer from 1 to 100. Inembodiments, z4 is independently an integer from 1 to 150. Inembodiments, z4 is independently an integer from 1 to 200. Inembodiments, z4 is independently an integer from 1 to 250. Inembodiments, z4 is independently an integer from 1 to 300. Inembodiments, z4 is independently an integer from 1 to 350. Inembodiments, z4 is independently an integer from 1 to 400. Inembodiments, z4 is independently an integer from 1 to 450. Inembodiments, z4 is independently an integer from 1 to 500. Inembodiments, z4 is independently an integer from 1 to 550. Inembodiments, z4 is independently an integer from 1 to 600. Inembodiments, z4 is independently an integer from 1 to 650. Inembodiments, z4 is independently an integer from 1 to 700. Inembodiments, z4 is independently an integer from 1 to 750. Inembodiments, z4 is independently an integer from 1 to 800. Inembodiments, z4 is independently an integer from 1 to 850. Inembodiments, z4 is independently an integer from 1 to 900. Inembodiments, z4 is independently an integer from 1 to 950. Inembodiments, z4 is independently an integer from 1 to 1000. Inembodiments, z4 is independently an integer from 1 to 1500. Inembodiments, z4 is independently an integer from 1 to 2000. Inembodiments, z4 is independently an integer from 1 to 2500. Inembodiments, z4 is independently an integer from 1 to 3000. Inembodiments, z4 is independently an integer from 1 to 3500. Inembodiments, z4 is independently an integer from 1 to 4000. Inembodiments, z4 is independently an integer from 1 to 4500. Inembodiments, z4 is independently an integer from 1 to 5000.

In some embodiments, each of the plurality of bioconjugate reactivemoieties include an amine moiety, aldehyde moiety, alkyne moiety, azidemoiety, carboxylic acid moiety, dibenzocyclooctyne (DBCO) moiety,tetrazine moiety, epoxy moiety, isocyanate moiety, furan moiety,maleimide moiety, thiol moiety, or transcyclooctene (TCO) moiety. Insome embodiments, each of the plurality of bioconjugate reactivemoieties include an amine moiety, azide moiety, dibenzocyclooctyne(DBCO) moiety, epoxy moiety, or isocyanate moiety. In embodiments, eachof the plurality of bioconjugate reactive moieties include an aminemoiety, azide moiety, alkyne moiety, dibenzocyclooctyne (DBCO) moiety,epoxy moiety, or isocyanate moiety. In embodiments, the bioconjugatereactive moiety is an azido moiety. In embodiments, the firstbioconjugate reactive moiety is an amine moiety, aldehyde moiety, alkynemoiety, azide moiety, carboxylic acid moiety, dibenzocyclooctyne (DBCO)moiety, tetrazine moiety, epoxy moiety, isocyanate moiety, furan moiety,maleimide moiety, thiol moiety, or transcyclooctene (TCO) moiety. Inembodiments, the second bioconjugate reactive moiety is an amine moiety,aldehyde moiety, alkyne moiety, azide moiety, carboxylic acid moiety,dibenzocyclooctyne (DBCO) moiety, tetrazine moiety, epoxy moiety,isocyanate moiety, furan moiety, maleimide moiety, thiol moiety, ortranscyclooctene (TCO) moiety. In embodiments, the first and the secondbioconjugate reactive moieties are different. In embodiments, the firstand the second bioconjugate reactive moieties are reactive with eachother (e.g., an azide moiety and an DBCO moiety) to form a bioconjugatelinker.

In embodiments, the particle polymer includes a copolymer of two or moreof the following polymerizable monomers, wherein at least one of thepolymerizable monomers includes a bioconjugate reactive moiety:polyacrylamide (AAm), poly-N-isopropylacrylamide, polyN-isopropylpolyacrylamide, sulfobetaine acrylate (SBA), carboxybetaineacrylate (CBA), phosphorylcholine acrylate (PCA), sulfobetainemethacrylate (SBMA), carboxybetaine methacrylate (CBMA),phosphorylcholine methacrylate (PCMA), polyethylene glycol acrylate,methacrylate, N-vinyl pyrrolidone, polyethylene glycol(PEG)-thiol/PEG-acrylate, acrylamide/N,N′-bis(acryloyl)cystamine (BACy),PEG/polypropylene oxide (PPO), polyacrylic acid, poly(hydroxyethylmethacrylate) (PHEMA), poly(methyl methacrylate) (PMMA),poly(N-isopropylacrylamide) (PNIPAAm), poly(lactic acid) (PLA),poly(lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL),poly(vinylsulfonic acid) (PVSA), poly(L-aspartic acid), poly(L-glutamicacid), polylysine, agar, agarose, alginate, heparin, alginate sulfate,dextran sulfate, hyaluronan, pectin, carrageenan, gelatin, chitosan,cellulose, collagen, glicydyl methacrylate (GMA), glicydyl methacrylate(GMA) azide, hydroxyethylmethacrylate (HEMA), hydroxyethylacrylate(HEA), hydroxypropylmethacrylate (HPMA), polyethylene glycolmethacrylate (PEGMA), polyethylene glycol acrylate (PEGA), and/orisocyanatoethyl methacrylate (IEM).

In embodiments, the oligonucleotide moiety (alternatively referred toherein as primer or polynucleotide primer) is covalently attached to thepolymer. In embodiments, the 5′ end of the oligonucleotide moietycontains a functional group that is tethered to the polymer (i.e., theparticle shell polymer or the polymeric particle). Non-limiting examplesof covalent attachment include amine-modified oligonucleotide moietiesreacting with epoxy or isothiocyanate groups on the polymer,succinylated oligonucleotide moieties reacting with aminophenyl oraminopropyl functional groups on the polymer, dibenzocycloctyne-modifiedoligonucleotide moieties reacting with azide functional groups on theparticle polymer (or vice versa), trans-cyclooctyne-modifiedoligonucleotide moieties reacting with tetrazine or methyl tetrazinegroups on the polymer (or vice versa), disulfide modifiedoligonucleotide moieties reacting with mercapto-functional groups on thepolymer, amine-functionalized oligonucleotide moieties reacting withcarboxylic acid groups on the polymer via1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC)chemistry, thiol-modified oligonucleotide moieties attaching to apolymer via a disulfide bond or maleimide linkage, alkyne-modifiedoligonucleotide moieties attaching to a polymer via copper-catalyzedclick reactions to azide functional groups on the polymer, andacrydite-modified oligonucleotide moieties polymerizing with freeacrylic acid monomers on the polymer to form polyacrylamide or reactingwith thiol groups on the polymer. In embodiments, the oligonucleotidemoiety is attached to the polymer through electrostatic binding. Forexample, the negatively charged phosphate backbone of the primer may bebound electrostatically to positively charged monomers in the polymer.

In embodiments, each particle includes multiple copies of one or moreoligonucleotide moieties. In embodiments, each particle includesmultiple copies oligonucleotide moieties having the same sequence. Inembodiments, the one or more oligonucleotide moieties include at leasttwo different primers attached to the polymer (e.g., a forward and areverse primer), each of which may be present in multiple copies. Inembodiments, about or at most at most about 50%, 40%, 30%, 25%, 20%,15%, 10%, 5%, or less of the monomers in the polymer of each particleare attached to a copy of the oligonucleotide moiety. In embodiments,about 1-25%, about 2-20%, about 3-15%, about 4-14%, or about 5-12% ofthe monomers in the polymer of each particle are attached to a copy ofthe oligonucleotide moiety, or a number or a range between any two ofthese values. In embodiments, about 5-10% of the monomers in the polymerof each particle are attached to a copy of the oligonucleotide moiety.In embodiments, two different oligonucleotide moieties are attached tothe particle (e.g., a forward and a reverse primer), which facilitatesgenerating multiple amplification products from the first extensionproduct or a complement thereof.

In embodiments, each of the particles include oligonucleotide moietiessubstantially identical to all the particles in the array. Inembodiments, each of the particles include at least two species ofoligonucleotide moieties that are substantially identical to all theparticles in the array. In embodiments, each of the particles comprisesubstantially the same oligonucleotide moieties (e.g., a firstpopulation of oligonucleotide moieties and a second population ofoligonucleotide moieties). In embodiments, each of the particlescomprise at least two species of substantially the same oligonucleotidemoieties (i.e., the same sequences). In embodiments, each particleincludes a plurality of P7 or P5 nucleic acid sequences or complementarysequences thereof (i.e., P5′ or P7′). The P5 and P7 adapter sequencesare described in U.S. Patent Publication No. 2011/0059865 A1, which isincorporated herein by reference in its entirety. The terms P5 and P7may be used when referring to amplification primers, e.g., universalprimers. The terms P5′ (P5 prime) and P7′ (P7 prime) refer to thecomplement of P5 and P7, respectively. In embodiments, each particleincludes a first plurality of a platform primer sequence and a secondplurality of a differing platform primer sequence. In embodiments, theplatform primer sequence is used during amplification reactions (e.g.,solid phase amplification). In embodiments, each particle includesoligonucleotide moieties capable of annealing to an adapter of a librarynucleic acid molecule. The term “library” merely refers to a collectionor plurality of template nucleic acid molecules which share commonsequences at their 5′ ends (e.g., the first end) and common sequences attheir 3′ ends (e.g., the second end). The term “adapter” as used hereinrefers to any linear oligonucleotide that can be ligated to a nucleicacid molecule, thereby generating nucleic acid products that can besequenced on a sequencing platform (e.g., an Illumina or SingularGenomics' G4™ sequencing platform). In embodiments, adapters include tworeverse complementary oligonucleotides forming a double-strandedstructure. In embodiments, an adapter includes two oligonucleotides thatare complementary at one portion and mismatched at another portion,forming a Y-shaped or fork-shaped adapter that is double stranded at thecomplementary portion and has two overhangs at the mismatched portion.Since Y-shaped adapters have a complementary, double-stranded region,they can be considered a special form of double-stranded adapters. Whenthis disclosure contrasts Y-shaped adapters and double strandedadapters, the term “double-stranded adapter” or “blunt-ended” is used torefer to an adapter having two strands that are fully complementary,substantially (e.g., more than 90% or 95%) complementary, or partiallycomplementary. In embodiments, adapters include sequences that bind tosequencing primers. In embodiments, adapters include sequences that bindto immobilized oligonucleotides (e.g., P7 and P5 sequences) or reversecomplements thereof. In embodiments, the adapter is substantiallynon-complementary to the 3′ end or the 5′ end of any targetpolynucleotide present in the sample. In embodiments, the adapter caninclude a sequence that is substantially identical, or substantiallycomplementary, to at least a portion of a primer, for example auniversal primer. In embodiments, the adapter can include an indexsequence (also referred to as barcode or tag) to assist with downstreamerror correction, identification or sequencing. In embodiments, each ofthe particles include at least two populations of substantially the sameoligonucleotide moieties.

In some embodiments, the oligonucleotide moiety is about 5 to about 50nucleotides in length. In some embodiments, the oligonucleotide moietyis about 5 to about 40 nucleotides in length. In some embodiments, theoligonucleotide moiety is about 10 to about 45 nucleotides in length. Insome embodiments, the oligonucleotide moiety is about 15 to about 40nucleotides in length. In some embodiments, the oligonucleotide moietyis about 20 to about 35 nucleotides in length. In some embodiments, theoligonucleotide moiety is about 20 to about 30 nucleotides in length. Insome embodiments, the oligonucleotide moiety is about 25 to about 30nucleotides in length. In embodiments, the oligonucleotide moiety isabout 25 to about 35 nucleotides in length. In embodiments, theoligonucleotide moiety is about 30 to about 50 nucleotides in length. Inembodiments, the oligonucleotide moiety is about 30 to about 75nucleotides in length. In embodiments, the oligonucleotide moiety isabout 50 to about 150 nucleotides in length. In embodiments, theoligonucleotide moiety is about 75 to about 200 nucleotides in length.In embodiments, the oligonucleotide moiety is a capture oligonucleotide,wherein the oligonucleotide is capable of hybridizing to a commonsequence in a library of nucleic acid molecules. In embodiments, theoligonucleotide is capable of hybridizing to a common sequence (e.g., asequence described in U.S. Patent Publication 2016/0256846, which isincorporated herein by reference, for example SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO: 5, or SEQ ID NO: 11 of U.S. Patent Publication2016/0256846).

In embodiments, the oligonucleotide moiety includes spacer nucleotides.Including spacer nucleotides in the linker puts the targetpolynucleotide in an environment having a greater resemblance to freesolution. This can be beneficial, for example, in enzyme-mediatedreactions such as sequencing-by-synthesis. It is believed that suchreactions suffer less steric hindrance issues that can occur when thepolynucleotide is directly attached to the particle or is attachedthrough a very short linker (e.g., a linker comprising about 1 to 3carbon atoms). Spacer nucleotides form part of the oligonucleotidemoiety but do not participate in any reaction carried out on or with theoligonucleotide (e.g., a hybridization or amplification reaction). Inembodiments, the spacer nucleotides include 1 to 20 nucleotides. Inembodiments, the linker includes 10 spacer nucleotides. In embodiments,the linker includes 12 spacer nucleotides. In embodiments, the linkerincludes 15 spacer nucleotides. It is preferred to use polyT spacers,although other nucleotides and combinations thereof can be used. Inembodiments, the linker includes 10, 11, 12, 13, 14, or 15 T spacernucleotides. In embodiments, the linker includes 12 T spacernucleotides. Spacer nucleotides are typically included at the 5′ ends ofoligonucleotide which are attached to the particle. Attachment can beachieved via a phosphorothioate present at the 5′ end of theoligonucleotide, an azide moiety, a dibenzocyclooctyne (DBCO) moiety, orany other bioconjugate reactive moiety (e.g., a bioconjugate moiety asdescribed herein). The linker may be a carbon-containing chain such asthose of formula —(CH₂)n- wherein “n” is from 1 to about 1000. However,a variety of other linkers may be used so long as the linkers are stableunder conditions used in DNA sequencing. In embodiments, the linkerincludes polyethylene glycol (PEG) having a general formula of—(CH₂—CH₂—O)m-, wherein m is from about 1 to 500.

In embodiments, the linker, or the oligonucleotides (e.g., primers)include a cleavable site. A cleavage site is a site which allowscontrolled cleavage of the immobilized polynucleotide strand (e.g., thelinker, the primer, or the polynucleotide) by chemical, enzymatic orphotochemical means. Any suitable enzymatic, chemical, or photochemicalcleavage reaction may be used to cleave the cleavage site. The cleavagereaction may result in removal of a part or the whole of the strandbeing cleaved. Suitable cleavage means include, for example, restrictionenzyme digestion, in which case the cleavage site is an appropriaterestriction site for the enzyme which directs cleavage of one or bothstrands of a duplex template; RNase digestion or chemical cleavage of abond between a deoxyribonucleotide and a ribonucleotide, in which casethe cleavage site may include one or more ribonucleotides; chemicalreduction of a disulfide linkage with a reducing agent (e.g., THPP orTCEP), in which case the cleavage site should include an appropriatedisulfide linkage; chemical cleavage of a diol linkage with periodate,in which case the cleavage site should include a diol linkage;generation of an abasic site and subsequent hydrolysis, etc. Inembodiments, the cleavage site is included in the oligonucleotide (e.g.,within the oligonucleotide sequence of the primer). In embodiments, thelinker or the oligonucleotide includes a diol linkage which permitscleavage by treatment with periodate (e.g., sodium periodate). It willbe appreciated that more than one diol can be included at the cleavagesite. One or more diol units may be incorporated into a polynucleotideusing standard methods for automated chemical DNA synthesis.Oligonucleotide nucleotide primers including one or more diol linkerscan be conveniently prepared by chemical synthesis. The diol linker iscleaved by treatment with any substance which promotes cleavage of thediol (e.g., a diol-cleaving agent). In embodiments, the diol-cleavingagent is periodate, e.g., aqueous sodium periodate (NaIO₄). Followingtreatment with the diol-cleaving agent (e.g., periodate) to cleave thediol, the cleaved product may be treated with a “capping agent” in orderto neutralize reactive species generated in the cleavage reaction.Suitable capping agents for this purpose include amines, e.g.,ethanolamine or propanolamine. In embodiments, cleavage may beaccomplished by using a modified nucleotide as the cleavable site (e.g.,uracil, 8oxoG, 5-mC, 5-hmC) that is removed or nicked via acorresponding DNA glycosylase, endonuclease, or combination thereof.

In embodiments, each of the particle-immobilized oligonucleotides (e.g.,immobilized primers) is about 5 to about 25 nucleotides in length. Inembodiments, each of the particle-immobilized oligonucleotides (e.g.,immobilized primers) is about 10 to about 40 nucleotides in length. Inembodiments, each of the particle-immobilized oligonucleotides (e.g.,immobilized primers) is about 5 to about 100 nucleotides in length. Inembodiments, each of the particle-immobilized oligonucleotides (e.g.,immobilized primers) is about 20 to 200 nucleotides in length. Inembodiments, each of the particle-immobilized oligonucleotides (e.g.,immobilized primers) about or at least about 5, 6, 7, 8, 9, 10, 12, 15,18, 20, 25, 30, 35, 40, 50 or more nucleotides in length. Inembodiments, one or more particle-immobilized oligonucleotides includeblocking groups at their 3′ ends that prevent polymerase extension. Ablocking moiety prevents formation of a covalent bond between the 3′hydroxyl moiety of the nucleotide and the 5′ phosphate of anothernucleotide. In embodiments, the 3′ modification is a 3′-phosphatemodification includes a 3′ phosphate moiety, which is removed by a PNKenzyme.

In embodiments, the oligonucleotide moiety includes one or morephosphorothioate nucleotides. In embodiments, the oligonucleotide moietyincludes a plurality of phosphorothioate nucleotides. In embodiments,about or at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or about100% of the nucleotides in the oligonucleotide moiety arephosphorothioate nucleotides. In embodiments, most of the nucleotides inthe oligonucleotide moiety are phosphorothioate nucleotides. Inembodiments, all of the nucleotides in the oligonucleotide moiety arephosphorothioate nucleotides. In embodiments, none of the nucleotides inthe oligonucleotide moiety are phosphorothioate nucleotides.

In some embodiments, the oligonucleotide moiety is capable ofhybridizing to a complementary sequence of a template nucleic acid. Inembodiments, the oligonucleotide moiety includes DNA. In embodiments,the oligonucleotide moiety includes RNA. In embodiments, theoligonucleotide moiety is DNA. In embodiments, the oligonucleotidemoiety is RNA. In embodiments, the oligonucleotide moiety includes asingle-stranded DNA. In embodiments, the oligonucleotide moiety includesa single-stranded RNA. In embodiments, the oligonucleotide moiety is asingle-stranded DNA. In embodiments, the oligonucleotide moiety is asingle-stranded RNA. In embodiments, the oligonucleotide moiety is anucleic acid sequence complementary to a target polynucleotide (e.g.,complementary to a common adapter sequence of the targetpolynucleotide).

In some embodiments, the particle includes a plurality of bioconjugatereactive moieties. In embodiments, the particle includes a plurality ofazide moieties, alkyne moieties, dibenzocyclooctyne (DBCO) moieties,epoxy moieties, or isocyanate moieties. In some embodiments, theparticle includes a plurality of oligonucleotide moieties (e.g., ssDNAmoieties).

In embodiments, the compositions described herein (e.g., the solidsupport and/or the particle) do not includepoly(N-(5-azidoacetamidylpentyl)acrylamide-co-acrylamide (PAZAM).

In an aspect is provided a nucleic acid sequencing device, including: astage configured to hold an array as described herein; an array asdescribed herein, including embodiments; and a detector for obtainingsequencing data. In some embodiments, the detector is an imagingdetector, such as a CCD, EMCCD, or s-CMOS detector.

In an aspect is provided a nucleic acid sequencing device, including: astage configured to hold an array or solid support as described herein,including embodiments; an array or solid support as described herein,including embodiments; and a detector for obtaining sequencing data. Insome embodiments, the detector is an imaging detector, such as a CCD,EMCCD, or s-CMOS detector.

The term “nucleic acid sequencing device” means an integrated system ofone or more chambers, ports, and channels that are interconnected and influid communication and designed for carrying out an analytical reactionor process, either alone or in cooperation with an appliance orinstrument that provides support functions, such as sample introduction,fluid and/or reagent driving means, temperature control, detectionsystems, data collection and/or integration systems, for the purpose ofdetermining the nucleic acid sequence of a template polynucleotide.Nucleic acid sequencing devices may further include valves, pumps, andspecialized functional coatings on interior walls. Nucleic acidsequencing devices may include a receiving unit, or platen, that orientsthe flow cell such that a maximal surface area of the flow cell isavailable to be exposed to an optical lens. Other nucleic acidsequencing devices include those provided by Singular Genomics™ such asthe G4™ sequencing platform, Illumina™, Inc. (e.g., HiSeq™, MiSeq™,NextSeq™, or NovaSeq™ systems), Life Technologies™ (e.g., ABI PRISM™, orSOLiD™ systems), Pacific Biosciences (e.g., systems using SMRT™Technology such as the Sequel™ or RS II™ systems), or Qiagen (e.g.,Genereader™ system). Nucleic acid sequencing devices may further includefluidic reservoirs (e.g., bottles), valves, pressure sources, pumps,sensors, control systems, valves, pumps, and specialized functionalcoatings on interior walls. In embodiments, the device includes aplurality of a sequencing reagent reservoirs and a plurality ofclustering reagent reservoirs. In embodiments, the clustering reagentreservoir includes amplification reagents (e.g., an aqueous buffercontaining enzymes, salts, and nucleotides, denaturants, crowdingagents, etc.) In embodiments, the reservoirs include sequencing reagents(such as an aqueous buffer containing enzymes, salts, and nucleotides);a wash solution (an aqueous buffer); a cleave solution (an aqueousbuffer containing a cleaving agent, such as a reducing agent); or acleaning solution (a dilute bleach solution, dilute NaOH solution,dilute HCl solution, dilute antibacterial solution, or water). The fluidof each of the reservoirs can vary. The fluid can be, for example, anaqueous solution which may contain buffers (e.g., saline-sodium citrate(SSC), ascorbic acid, tris(hydroxymethyl)aminomethane or “Tris”),aqueous salts (e.g., KCl or (NH₄)₂SO₄)), nucleotides, polymerases,cleaving agent (e.g., tri-n-butyl-phosphine, triphenyl phosphine and itssulfonated versions (i.e., tris(3-sulfophenyl)-phosphine, TPPTS), andtri(carboxyethyl)phosphine (TCEP) and its salts, cleaving agentscavenger compounds (e.g., 2′-Dithiobisethanamine or11-Azido-3,6,9-trioxaundecane-1-amine), chelating agents (e.g., EDTA),detergents, surfactants, crowding agents, or stabilizers (e.g., PEG,Tween, BSA). Non-limited examples of reservoirs include cartridges,pouches, vials, containers, and eppendorf tubes. In embodiments, thedevice is configured to perform fluorescent imaging. In embodiments, thedevice includes one or more light sources (e.g., one or more lasers). Inembodiments, the illuminator or light source is a radiation source(i.e., an origin or generator of propagated electromagnetic energy)providing incident light to the sample. A radiation source can includean illumination source producing electromagnetic radiation in theultraviolet (UV) range (about 200 to 390 nm), visible (VIS) range (about390 to 770 nm), or infrared (IR) range (about 0.77 to 25 microns), orother range of the electromagnetic spectrum. In embodiments, theilluminator or light source is a lamp such as an arc lamp or quartzhalogen lamp. In embodiments, the illuminator or light source is acoherent light source. In embodiments, the light source is a laser, LED(light emitting diode), a mercury or tungsten lamp, or asuper-continuous diode. In embodiments, the light source providesexcitation beams having a wavelength between 200 nm to 1500 nm. Inembodiments, the laser provides excitation beams having a wavelength of405 nm, 470 nm, 488 nm, 514 nm, 520 nm, 532 nm, 561 nm, 633 nm, 639 nm,640 nm, 800 nm, 808 nm, 912 nm, 1024 nm, or 1500 nm. In embodiments, theilluminator or light source is a light-emitting diode (LED). The LED canbe, for example, an Organic Light Emitting Diode (OLED), a Thin FilmElectroluminescent Device (TFELD), or a Quantum dot based inorganicorganic LED. The LED can include a phosphorescent OLED (PHOLED). Inembodiments, the nucleic acid sequencing device includes an imagingsystem (e.g., an imaging system as described herein). The imaging systemcapable of exciting one or more of the identifiable labels (e.g., afluorescent label) linked to a nucleotide and thereafter obtain imagedata for the identifiable labels. The image data (e.g., detection data)may be analyzed by another component within the device. The imagingsystem may include a system described herein and may include afluorescence spectrophotometer including an objective lens and/or asolid-state imaging device. The solid-state imaging device may include acharge coupled device (CCD) and/or a complementary metal oxidesemiconductor (CMOS). The system may also include circuitry andprocessors, including systems using microcontrollers, reducedinstruction set computers (RISC), application specific integratedcircuits (ASICs), field programmable gate array (FPGAs), logic circuits,and any other circuit or processor capable of executing functionsdescribed herein. The set of instructions may be in the form of asoftware program. As used herein, the terms “software” and “firmware”are interchangeable, and include any computer program stored in memoryfor execution by a computer, including RAM memory, ROM memory, EPROMmemory, EEPROM memory, and non-volatile RAM (NVRAM) memory. Inembodiments, the device includes a thermal control assembly useful tocontrol the temperature of the reagents.

In an aspect is provided a flow cell including a particle as describedherein, wherein the particle is within a well of the flow cell.

In an aspect is provided a kit, including the array as described herein.In an aspect is provided a kit, including the solid support as describedherein. Generally, the kit includes one or more containers providing acomposition and one or more additional reagents (e.g., a buffer suitablefor polynucleotide extension). The kit may also include a templatenucleic acid (DNA and/or RNA), one or more primer polynucleotides,nucleoside triphosphates (including, e.g., deoxyribonucleotides,ribonucleotides, particles, labeled nucleotides, and/or modifiednucleotides), buffers, salts, and/or labels (e.g., fluorophores). Inembodiments, the kit includes an array with particles already loadedinto the wells. In embodiments, the particles are in a container. Inembodiments, the particles are in aqueous suspension or as a powderwithin the container. The container may be a storage device or otherreadily usable vessel capable of storing and protecting the particles.The kit may also include a flow cell. In embodiments, kit includes thesolid support and a flow cell carrier (e.g., a flow cell carrier asdescribed in US 2021/0190668, which is incorporated herein by referencefor all purposes).

In an aspect is provided a kit, including the plurality of particles asdescribed herein. Generally, the kit includes one or more containersproviding a composition and one or more additional reagents (e.g., abuffer suitable for polynucleotide extension).

In embodiments, the kit includes a sequencing polymerase, and one ormore amplification polymerases. In embodiments, the sequencingpolymerase is capable of incorporating modified nucleotides. Inembodiments, the polymerase is a DNA polymerase. In embodiments, the DNApolymerase is a Pol I DNA polymerase, Pol II DNA polymerase, Pol III DNApolymerase, Pol IV DNA polymerase, Pol V DNA polymerase, Pol β DNApolymerase, Pol μ DNA polymerase, Pol λ DNA polymerase, Pol σ DNApolymerase, Pol α DNA polymerase, Pol δ DNA polymerase, Pol ε DNApolymerase, Pol η DNA polymerase, Pol ι DNA polymerase, Pol κ DNApolymerase, Pol ζ DNA polymerase, Pol γ DNA polymerase, Pol θ DNApolymerase, Pol υ DNA polymerase, or a thermophilic nucleic acidpolymerase (e.g., Therminator γ, 9° N polymerase (exo-), Therminator II,Therminator III, or Therminator IX). In embodiments, the DNA polymeraseis a thermophilic nucleic acid polymerase. In embodiments, the DNApolymerase is a modified archaeal DNA polymerase. In embodiments, thepolymerase is a reverse transcriptase. In embodiments, the polymerase isa mutant P. abyssi polymerase (e.g., such as a mutant P. abyssipolymerase described in WO 2018/148723 or WO 2020/056044, each of whichare incorporated herein by reference for all purposes). In embodiments,the kit includes a strand-displacing polymerase. In embodiments, the kitincludes a strand-displacing polymerase, such as a phi29 polymerase,phi29 mutant polymerase or a thermostable phi29 mutant polymerase.

In embodiments, the kit includes a buffered solution. Typically, thebuffered solutions contemplated herein are made from a weak acid and itsconjugate base or a weak base and its conjugate acid. For example,sodium acetate and acetic acid are buffer agents that can be used toform an acetate buffer. Other examples of buffer agents that can be usedto make buffered solutions include, but are not limited to, Tris,bicine, tricine, HEPES, TES, MOPS, MOPSO and PIPES. Additionally, otherbuffer agents that can be used in enzyme reactions, hybridizationreactions, and detection reactions are known in the art. In embodiments,the buffered solution can include Tris. With respect to the embodimentsdescribed herein, the pH of the buffered solution can be modulated topermit any of the described reactions. In some embodiments, the bufferedsolution can have a pH greater than pH 7.0, greater than pH 7.5, greaterthan pH 8.0, greater than pH 8.5, greater than pH 9.0, greater than pH9.5, greater than pH 10, greater than pH 10.5, greater than pH 11.0, orgreater than pH 11.5. In other embodiments, the buffered solution canhave a pH ranging, for example, from about pH 6 to about pH 9, fromabout pH 8 to about pH 10, or from about pH 7 to about pH 9. Inembodiments, the buffered solution can comprise one or more divalentcations. Examples of divalent cations can include, but are not limitedto, Mg²⁺, Mn²⁺, Zn²⁺, and Ca²⁺. In embodiments, the buffered solutioncan contain one or more divalent cations at a concentration sufficientto permit hybridization of a nucleic acid.

As used herein, the term “kit” refers to any delivery system fordelivering materials. In the context of reaction assays, such deliverysystems include systems that allow for the storage, transport, ordelivery of reaction reagents (e.g., oligonucleotides, enzymes, etc. inthe appropriate containers) and/or supporting materials (e.g., buffers,written instructions for performing the assay, etc.) from one locationto another. For example, kits include one or more enclosures (e.g.,boxes) containing the relevant reaction reagents and/or supportingmaterials. As used herein, the term “fragmented kit” refers to adelivery system comprising two or more separate containers that eachcontain a subportion of the total kit components. The containers may bedelivered to the intended recipient together or separately. For example,a first container may contain an enzyme for use in an assay, while asecond container contains oligonucleotides. In contrast, a “combinedkit” refers to a delivery system containing all of the components of areaction assay in a single container (e.g., in a single box housing eachof the desired components). The term “kit” includes both fragmented andcombined kits. In embodiments, the kit includes, without limitation,nucleic acid primers, probes, adapters, enzymes, and the like, and areeach packaged in a container, such as, without limitation, a vial, tubeor bottle, in a package suitable for commercial distribution, such as,without limitation, a box, a sealed pouch, a blister pack and a carton.The package typically contains a label or packaging insert indicatingthe uses of the packaged materials. As used herein, “packagingmaterials” includes any article used in the packaging for distributionof reagents in a kit, including without limitation containers, vials,tubes, bottles, pouches, blister packaging, labels, tags, instructionsheets and package inserts.

III. Methods

In an aspect is provided a method of amplifying a target polynucleotide,the method including contacting an array as described herein, includingembodiments, with a plurality of oligonucleotide moieties. Inembodiments, the array includes a solid support including a surface, thesurface comprising a plurality of wells separated from each other byinterstitial regions on the surface, wherein one or more wells includesa particle, wherein the particle includes a plurality of bioconjugatereactive moieties; and wherein there is at least one particle per well.In embodiments, each oligonucleotide moiety includes a bioconjugatereactive moiety that reacts and forms a bioconjugate linker thatcovalently links the oligonucleotide moiety to the particle. Inembodiments, the method includes contacting the array with a sampleincluding a target polynucleotide; and amplifying the targetpolynucleotide to produce an amplification product (e.g., an amplicon),wherein amplifying includes extension of the oligonucleotide moietyhybridized to the target polynucleotide.

In an aspect is provided a method of amplifying a target polynucleotide,the method including contacting an array, alternatively referred to as amultiwell container and/or solid support as described herein, includingembodiments, with a plurality of oligonucleotide moieties, andamplifying the target polynucleotide hybridized to the oligonucleotides.

In an aspect is provided a method of amplifying a target polynucleotide,the method including contacting an array or solid support as describedherein, including embodiments, with a plurality of oligonucleotidemoieties. In embodiments, the array includes a solid support including asurface, the surface comprising a plurality of wells separated from eachother by interstitial regions on the surface, wherein one or more wellsincludes a particle, wherein the particle includes a plurality ofbioconjugate reactive moieties; and wherein there is at least oneparticle per well. In embodiments, each oligonucleotide moiety includesa bioconjugate reactive moiety that reacts and forms a bioconjugatelinker that covalently links the oligonucleotide moiety to the particle.In embodiments, the method includes contacting the array with a sampleincluding a target polynucleotide; and amplifying the targetpolynucleotide to produce an amplification product (e.g., an amplicon),wherein amplifying includes extension of the oligonucleotide moietyhybridized to the target polynucleotide.

In embodiments, the oligonucleotide moiety includes a DBCO bioconjugatereactive moiety that reacts with an azide bioconjugate reactive moietyon the particle and forms a bioconjugate linker that covalently linksthe oligonucleotide moiety to the particle, for example according to thefollowing scheme:

The symbol

refers to the attachment point to the oligonucleotide moiety and theparticle polymer, respectively.

In an aspect is provided a method of amplifying a target polynucleotide,the method including contacting an array with a sample including atarget polynucleotide. In embodiments, the array includes a solidsupport including a surface, the surface comprising a plurality of wellsseparated from each other by interstitial regions on the surface,wherein one or more wells includes a particle, wherein the particleincludes a plurality of oligonucleotide moieties; and wherein there isat least one particle per well. In embodiments, the array includes asolid support as described herein. In embodiments the solid supportincludes a surface, the surface comprising a plurality of wellsseparated from each other by interstitial regions on the surface,wherein one or more wells includes a particle, wherein the particleincludes a plurality of oligonucleotide moieties; and wherein there isat least one particle per well. In embodiments, the method includesamplifying the target polynucleotide to produce an amplificationproduct, wherein amplifying includes extension of the oligonucleotidemoiety hybridized to the target polynucleotide. In embodiments,amplifying includes a plurality of cycles of strand denaturation, primerhybridization, and primer extension.

In an aspect is provided a method of amplifying a target polynucleotide.In embodiments, the method includes contacting the solid support asdescribed herein with a sample including a target polynucleotide; andamplifying the target polynucleotide to produce an amplificationproduct, wherein amplifying includes extension of the oligonucleotidemoiety hybridized to the target polynucleotide. In embodiments, thetarget polynucleotide includes a complementary sequence to theoligonucleotide covalently attached to the particle.

In embodiments, the method includes contacting a particle as describedherein with a sample including a target polynucleotide; and amplifyingthe target polynucleotide to produce an amplification productimmobilized to the particle, wherein amplifying includes extension ofthe oligonucleotide moiety hybridized to the target polynucleotide, andcontacting the solid support with the amplified product immobilized tothe particle. In embodiments, the target polynucleotide includes acomplementary sequence to the oligonucleotide covalently attached to theparticle. In embodiments, the method further includes detecting theamplification product (e.g., sequencing the amplification product).

In embodiments, amplifying includes bridge polymerase chain reaction(bPCR) amplification, solid-phase rolling circle amplification (RCA),solid-phase exponential rolling circle amplification (eRCA), solid-phaserecombinase polymerase amplification (RPA), solid-phase helicasedependent amplification (HDA), template walking amplification, oremulsion PCR on particles, or combinations of the methods. Inembodiments, amplifying includes a bridge polymerase chain reactionamplification. In embodiments, amplifying includes a thermal bridgepolymerase chain reaction (t-bPCR) amplification. In embodiments,amplifying includes a chemical bridge polymerase chain reaction (c-bPCR)amplification. Chemical bridge polymerase chain reactions includefluidically cycling a denaturant (e.g., formamide) and maintaining thetemperature within a narrow temperature range (e.g., +/−5° C.). Incontrast, thermal bridge polymerase chain reactions include thermallycycling between high temperatures (e.g., 85° C.-95° C.) and lowtemperatures (e.g., 60° C.-70° C.). Thermal bridge polymerase chainreactions may also include a denaturant, typically at a much lowerconcentration than traditional chemical bridge polymerase chainreactions. In embodiments, amplifying includes generating adouble-stranded amplification product.

A sample can be any specimen that is isolated or obtained from a subjector part thereof. A sample can be any specimen that is isolated orobtained from multiple subjects. Non-limiting examples of specimensinclude fluid or tissue from a subject, including, without limitation,blood or a blood product (e.g., serum, plasma, platelets, buffy coats,or the like), umbilical cord blood, chorionic villi, amniotic fluid,cerebrospinal fluid, spinal fluid, lavage fluid (e.g., lung, gastric,peritoneal, ductal, ear, arthroscopic), a biopsy sample, celocentesissample, cells (blood cells, lymphocytes, placental cells, stem cells,bone marrow derived cells, embryo or fetal cells) or parts thereof(e.g., mitochondrial, nucleus, extracts, or the like), urine, feces,sputum, saliva, nasal mucous, prostate fluid, lavage, semen, lymphaticfluid, bile, tears, sweat, breast milk, breast fluid, the like orcombinations thereof. Non-limiting examples of tissues include organtissues (e.g., liver, kidney, lung, thymus, adrenals, skin, bladder,reproductive organs, intestine, colon, spleen, brain, the like or partsthereof), epithelial tissue, hair, hair follicles, ducts, canals, bone,eye, nose, mouth, throat, ear, nails, the like, parts thereof orcombinations thereof. A sample may comprise cells or tissues that arenormal, healthy, diseased (e.g., infected), and/or cancerous (e.g.,cancer cells). A sample obtained from a subject may comprise cells orcellular material (e.g., nucleic acids) of multiple organisms (e.g.,virus nucleic acid, fetal nucleic acid, bacterial nucleic acid, parasitenucleic acid). A sample may include a cell and RNA transcripts. A samplecan comprise nucleic acids obtained from one or more subjects. In someembodiments, a sample comprises nucleic acid obtained from a singlesubject. A subject can be any living or non-living organism, includingbut not limited to a human, non-human animal, plant, bacterium, fungus,virus, or protist. A subject may be any age (e.g., an embryo, a fetus,infant, child, adult). A subject can be of any sex (e.g., male, female,or combination thereof). A subject may be pregnant. In some embodiments,a subject is a mammal. In some embodiments, a subject is a plant. Insome embodiments, a subject is a human subject. A subject can be apatient (e.g., a human patient). In some embodiments, a subject issuspected of having a genetic variation or a disease or conditionassociated with a genetic variation.

It will be appreciated that any of the amplification methodologiesdescribed herein or known in the art can be utilized with universal ortarget-specific primers to amplify the target polynucleotide. Suitablemethods for amplification include, but are not limited to, thepolymerase chain reaction (PCR), strand displacement amplification(SDA), transcription mediated amplification (TMA) and nucleic acidsequence-based amplification (NASBA), for example, as described in U.S.Pat. No. 8,003,354, which is incorporated herein by reference in itsentirety. The above amplification methods can be employed to amplify oneor more nucleic acids of interest. Additional examples of amplificationprocesses include, but are not limited to, bridge-PCR, recombinasepolymerase amplification (RPA), loop-mediated isothermal amplification(LAMP), rolling circle amplification (RCA), strand displacementamplification, RCA with exponential strand displacement amplification.In embodiments, amplification comprises an isothermal amplificationreaction. In embodiments, amplification comprises bridge amplification.In general, bridge amplification uses repeated steps of annealing ofprimers to templates, primer extension, and separation of extendedprimers from templates. Because primers are attached within the corepolymer, the extension products released upon separation from an initialtemplate is also attached within the core. The 3′ end of anamplification product is then permitted to anneal to a nearby reverseprimer that is also attached within the core, forming a “bridge”structure. The reverse primer is then extended to produce a furthertemplate molecule that can form another bridge. In embodiments, forwardand reverse primers hybridize to primer binding sites that are specificto a particular target nucleic acid. In embodiments, forward and reverseprimers hybridize to primer binding sites that have been added to, andare common among, target polynucleotides. Adding a primer binding siteto target nucleic acids can be accomplished by any suitable method,examples of which include the use of random primers having common 5′sequences and ligating adapter nucleotides that include the primerbinding site. Examples of additional clonal amplification techniquesinclude, but are not limited to, bridge PCR, solid-phase rolling circleamplification (RCA), solid-phase exponential rolling circleamplification, solid-phase recombinase polymerase amplification (RPA),solid-phase helicase dependent amplification (HDA), template walkingamplification, emulsion PCR on particles (beads), or combinations of theaforementioned methods. Optionally, during clonal amplification,additional solution-phase primers can be supplemented in the flow cellfor enabling or accelerating amplification.

In embodiments, amplifying includes contacting the plurality ofparticles with one or more reagents for amplifying the targetpolynucleotide. Examples of reagents include but are not limited topolymerase, buffer, and nucleotides (e.g., an amplification reactionmixture). In certain embodiments, the term “amplifying” refers to amethod that includes a polymerase chain reaction (PCR). Conditionsconducive to amplification (i.e., amplification conditions) are knownand often comprise at least a suitable polymerase, a suitable template,a suitable primer or set of primers, suitable nucleotides (e.g., dNTPs),a suitable buffer, and application of suitable annealing, hybridizationand/or extension times and temperatures. In embodiments, amplifyinggenerates an amplicon. In embodiments, an amplicon contains multiple,tandem copies of the circularized nucleic acid molecule of thecorresponding sample nucleic acid. The number of copies can be varied byappropriate modification of the amplification reaction including, forexample, varying the number of amplification cycles run, usingpolymerases of varying processivity in the amplification reaction and/orvarying the length of time that the amplification reaction is run, aswell as modification of other conditions known in the art to influenceamplification yield. Generally, the number of copies of a nucleic acidin an amplicon is at least 100, 200, 500, 1000, 2000, 3000, 4000, 5000,6000, 7000, 8000, 9000 and 10,000 copies, and can be varied depending onthe application. As disclosed herein, one form of an amplicon is as anucleic acid “ball” localized to the particle and/or well of the array.The number of copies of the nucleic acid can therefore provide a desiredsize of a nucleic acid “ball” or a sufficient number of copies forsubsequent analysis of the amplicon, e.g., sequencing.

In embodiments, the sample includes a plurality of targetpolynucleotides at a concentration selected such that a majority of theparticles in which the amplification occurs includes amplicons of onlyone original target polynucleotide. In embodiments, about or at leastabout 60%, 70%, 80%, 90%, 95%, or more of the particles in whichamplification occurs contains amplicons of only one original targetpolynucleotide.

In embodiments, the methods further include a step of separatingparticles that include amplicons (e.g., amplification product(s)) fromparticles that do not include amplicons. For example, particles that donot include amplicon, also referred to as “blank” particles, can beseparated from particles with amplicon by charge-based separation. SinceDNA carries a negative charge, the particles that contain amplifiedproducts will be significantly charged compared to the “blank”particles. The particles containing amplicons can be separated from the“blank” particles by an electric field. By choosing a relatively lowseeding ratio, e.g. <20%, or <10%, or <5%, the probability of havingmore than 1 template per particle (multiple seeding) can be greatlyreduced. The large fraction of unseeded “blank” particles can then beremoved by charge-based separation, resulting in nearly pure populationof single-seeded particles for monoclonal amplification. In embodiments,the methods comprise amplifying a target polynucleotide in solution,separating particles containing amplicons from “blank” particles, anddepositing the particles containing amplicons in a container (e.g., aflow cell) for sequencing. Alternatively, the particles may contain amagnetic core and may be separated by applying a magnetic field.

In embodiments, the methods further include repeating the contacting andamplifying steps using the separated particles that do not include anamplicon. In embodiments, the contacting is repeated with an aliquot ofthe same sample as in the original contacting, and particles from therepeated steps are pooled (e.g., in a container, such as a flow cell)prior to sequencing. In embodiments, repeating the contacting andamplifying steps does not involve separating particles that do notinclude an amplicon from those that do contain an amplicon.

In embodiments of the methods provided herein, arraying the particlesoccurs prior to contacting the particles with a sample that includes atarget polynucleotide. In other embodiments, arraying the particlesoccurs after contacting the particles with a sample that includes atarget polynucleotide. In other embodiments, arraying the particlesoccurs after amplifying the target polynucleotide.

In an aspect is provided a method of making an array of nucleic acids ona surface, the method including: a) providing a solid support includinga surface, the surface including a plurality of wells wherein the wellsare separated from each other by interstitial regions on the surface; b)providing a plurality of particles, wherein each particle includes aplurality of bioconjugate reactive moieties; c) arraying the particlesonto the surface; d) contacting the particles with a plurality ofoligonucleotide moieties, wherein each oligonucleotide moiety includes abioconjugate reactive moiety that reacts and forms a bioconjugate linkerthat covalently links the oligonucleotide moiety to the particle.

In an aspect is a method of making an array of nucleic acids on asurface, the method including: a) providing a solid support including asurface, the surface including a plurality of wells wherein the wellsare separated from each other by interstitial regions on the surface; b)providing a plurality of particles, wherein each particle includes aplurality of oligonucleotide moieties; c) arraying the particles ontothe surface; d) contacting the particles with a plurality of templatenucleic acid moieties, wherein a complementary sequence of the templatenucleic acid moieties hybridizes to the oligonucleotide moiety of theparticle. In embodiments, the average longest dimension of the particleis from about 150 nm to about 1,000 nm.

In an aspect is provided a method of making an array of nucleic acids ona surface, the method including: a) providing a solid support includinga surface, the surface including a plurality of wells wherein the wellsare separated from each other by interstitial regions on the surface andwherein the surface comprises a polymer layer and is substantially freeof oligonucleotide moieties; b) providing a plurality of particles; c)arraying the particles onto the surface; d) contacting the particleswith a plurality of template nucleic acid moieties, wherein acomplementary sequence of the template nucleic acid moieties hybridizesto the oligonucleotide moiety of the particle. In embodiments, theaverage longest dimension of the particle is from about 150 nm to about1,000 nm.

In another aspect is provided a method of making an array of templatenucleic acids, the method including: contacting a solid supportincluding two or more wells with a plurality of particles, wherein eachparticle includes a plurality of oligonucleotide moieties attached tothe particle via a bioconjugate linker; wherein the average longestdimension of the particle is from about 150 nm to about 1,000 nm; andcontacting the particles with a plurality of template nucleic acidmoieties, wherein a complementary sequence of the template nucleic acidmoieties hybridizes to the oligonucleotide moiety of the particle and isextended with a polymerase to form an array of template nucleic acids.

In embodiments of the methods provided herein, the contacting step isperformed under non-hybridizing conditions. In embodiments of themethods provided herein, the contacting step is performed undernon-hybridizing conditions initially, then the conditions are changed tohybridizing conditions. In embodiments of the methods provided herein,the contacting step is performed under hybridizing conditions initially,then the conditions are changed to non-hybridizing conditions. Ingeneral, contacting the sample under non-hybridizing conditions canfacilitate distribution of target polynucleotides within a polymericparticle prior to subsequent steps (e.g., amplification). Examples ofnon-hybridizing conditions include but are not limited to low salt, hightemperature, and/or presence of additives such as formamide. The precisenature of non-hybridizing conditions (e.g., the temperature, or theamounts of salt or formamide) will vary with factors such as the length,GC-content, or melting temperature (Tm) of primers (or thetarget-hybridizing portion thereof) present in the reaction. Inembodiments, primers are designed to have Tm's within 15, 10, 5, 3 orfewer degrees of one another. In embodiments, non-hybridizing conditionscomprises a temperature that is about or at least about 5, 10, 15, 20,or more degrees above the average Tm of primers in the reaction.

In embodiments, the method includes determining the nucleic acidsequence of the target polynucleotide. In embodiments, the methodfurther includes detecting the oligonucleotides, or extension productsor complements thereof. In embodiments, the method includes detectingthe template nucleic acid and/or determining the nucleic acid sequenceof the target polynucleotide. In embodiments, the molecule furtherincludes quantifying the target nucleic acid molecule or amplicons.Methods for quantifying a target polynucleotide or amplicon are wellknown to one of skilled in the art. For example, during amplification ofthe target nucleic acid, quantitative techniques such as real-timepolymerase chain reaction (RT-PCR) can be used to quantify the copynumber of target nucleic acid molecules present in the clonal object asdiscussed in Logan et al. Real-Time PCR: Current Technology andApplications, Caister Academic Press. (2009). RT-PCR follows the generalprinciple of polymerase chain reaction, however inclusion of detectionmolecules, such as non-specific fluorescent dyes that intercalate withany double-stranded DNA, or sequence-specific DNA probes consisting ofoligonucleotides that are labeled with a fluorescent reporter, whichpermits detection only after hybridization of the probe with itscomplementary DNA target, allows for the detection of nucleic acidformed during amplification. The rate of detectable molecules isproportional to the copy number of target nucleic acid molecules presentin the clonal object. Furthermore, quantifying the target nucleic acidmolecule or amplicons can be done following amplification using standardgel electrophoresis and/or Southern blot techniques, which are wellknown in the art.

In embodiments, the method further includes sequencing the amplificationproduct(s). Sequencing includes, for example, detecting a sequence ofsignals within the particle. Examples of sequencing include, but are notlimited to, sequencing by synthesis (SBS) processes in which reversiblyterminated nucleotides carrying fluorescent dyes are incorporated into agrowing strand, complementary to the target strand being sequenced. Inembodiments, the nucleotides are labeled with up to four uniquefluorescent dyes. In embodiments, the readout is accomplished byepifluorescence imaging. A variety of sequencing chemistries areavailable, non-limiting examples of which are described herein.

In an aspect is provided a method of sequencing a target polynucleotide,the method including contacting an array as described herein, includingembodiments, with a plurality of oligonucleotide moieties. Inembodiments, the array includes a solid support including a surface, thesurface comprising a plurality of wells separated from each other byinterstitial regions on the surface, wherein one or more wells includesa particle, wherein the particle includes a plurality of bioconjugatereactive moieties; and wherein there is at least one particle per well.In embodiments, each oligonucleotide moiety includes a bioconjugatereactive moiety that reacts and forms a bioconjugate linker thatcovalently links the oligonucleotide moiety to the particle. Inembodiments, the method includes contacting the array with a sampleincluding a target polynucleotide; and amplifying the targetpolynucleotide to produce an amplification product, wherein amplifyingincludes extension of the oligonucleotide moiety hybridized to thetarget polynucleotide. In embodiments, the method includes sequencingthe amplification product. The initiation point for a sequencingreaction may be provided by annealing of a sequencing primer to a targetpolynucleotide present at a feature of the array. In embodiments, aknown adapter sequence region that is present on a target nucleic acid,for example, as a result of an amplification reaction describedpreviously herein, can be used as a priming site for annealing of asequencing primer. In embodiments, a sequencing reaction includes stepsof hybridizing a sequencing primer to a single-stranded region of alinearized amplification product, sequentially incorporating one or morenucleotides into a nucleic acid strand complementary to the region ofamplified template strand to be sequenced, identifying the base presentin one or more of the incorporated nucleotide(s) and thereby determiningthe sequence of a region of the template strand.

In an aspect is provided a method of sequencing a target polynucleotide,the method including contacting an array with a sample including atarget polynucleotide. In embodiments, the array is as described herein.In embodiments the array includes a solid support including a surface,the surface including a plurality of wells separated from each other byinterstitial regions on the surface, wherein one or more wells includesa particle, wherein the particle includes a plurality of oligonucleotidemoieties; and wherein there is at least one particle per well. Inembodiments, the method includes amplifying the target polynucleotide toproduce an amplification product, wherein amplifying includes extensionof the oligonucleotide moiety hybridized to the target polynucleotide.In embodiments, the method includes sequencing the amplificationproduct.

In an aspect is provided a method of sequencing a templatepolynucleotide. In embodiments, the method includes contacting a solidsupport (e.g., a solid support as described herein) with a sampleincluding a template polynucleotide. In embodiments, the method includehybridizing the template polynucleotide to the oligonucleotide moiety.In embodiments, the method includes extending the oligonucleotide moietyto generate a complement of the template polynucleotide immobilized tothe particle. In embodiments, the method includes forming a plurality ofamplification products by subjecting the solid support to suitableamplification conditions (e.g., as described herein). In embodiments,the method includes contacting the immobilized template polynucleotide,or complement thereof, with a sequencing primer, and with a polymerase,incorporating one or more nucleotides into an extension strand. Inembodiments, the method includes detecting the one or more nucleotidesincorporated into the extension strand.

In embodiments, sequencing includes extending a sequencing primer toincorporate a nucleotide containing a detectable label that indicatesthe identity of a nucleotide in the target polynucleotide, detecting thedetectable label, and repeating the extending and detecting of steps. Inembodiments, the methods include sequencing one or more bases of atarget nucleic acid by extending a sequencing primer hybridized to atarget nucleic acid (e.g., an amplification product of a target nucleicacid). In embodiments, the sequencing includes sequencing-by-synthesis,sequencing by ligation, or pyrosequencing, and generates a sequencingread. In embodiments, the sequencing includes sequencing-by-binding andgenerates one or more sequencing reads.

In embodiments, generating a sequencing read includes executing aplurality of sequencing cycles, each cycle including extending thesequencing primer by incorporating a nucleotide or nucleotide analogueusing a polymerase and detecting a characteristic signature indicatingthat the nucleotide or nucleotide analogue has been incorporated.

In embodiments, the method includes sequencing the first and/or thesecond strand of a amplification product by extending a sequencingprimer hybridized thereto. A variety of sequencing methodologies can beused such as sequencing-by-synthesis (SBS), pyrosequencing, sequencingby ligation (SBL), or sequencing by hybridization (SBH). Pyrosequencingdetects the release of inorganic pyrophosphate (PPi) as particularnucleotides are incorporated into a nascent nucleic acid strand(Ronaghi, et al., Analytical Biochemistry 242(1), 84-9 (1996); Ronaghi,Genome Res. 11(1), 3-11 (2001); Ronaghi et al. Science 281(5375), 363(1998); U.S. Pat. Nos. 6,210,891; 6,258,568; and. 6,274,320, each ofwhich is incorporated herein by reference in its entirety). Inpyrosequencing, released PPi can be detected by being converted toadenosine triphosphate (ATP) by ATP sulfurylase, and the level of ATPgenerated can be detected via light produced by luciferase. In thismanner, the sequencing reaction can be monitored via a luminescencedetection system. In both SBL and SBH methods, target nucleic acids, andamplicons thereof, that are present at features of an array aresubjected to repeated cycles of oligonucleotide delivery and detection.SBL methods, include those described in Shendure et al. Science309:1728-1732 (2005); U.S. Pat. Nos. 5,599,675; and 5,750,341, each ofwhich is incorporated herein by reference in its entirety; and the SBHmethodologies are as described in Bains et al., Journal of TheoreticalBiology 135(3), 303-7 (1988); Drmanac et al., Nature Biotechnology 16,54-58 (1998); Fodor et al., Science 251(4995), 767-773 (1995); and WO1989/10977, each of which is incorporated herein by reference in itsentirety.

In SBS, extension of a nucleic acid primer along a nucleic acid templateis monitored to determine the sequence of nucleotides in the template.The underlying chemical process can be catalyzed by a polymerase,wherein fluorescently labeled nucleotides are added to a primer (therebyextending the primer) in a template dependent fashion such thatdetection of the order and type of nucleotides added to the primer canbe used to determine the sequence of the template. A plurality ofdifferent nucleic acid fragments that have been attached at differentlocations of an array can be subjected to an SBS technique underconditions where events occurring for different templates can bedistinguished due to their location in the array. In embodiments, thesequencing step includes annealing and extending a sequencing primer toincorporate a detectable label that indicates the identity of anucleotide in the target polynucleotide, detecting the detectable label,and repeating the extending and detecting steps. In embodiments, themethods include sequencing one or more bases of a target nucleic acid byextending a sequencing primer hybridized to a target nucleic acid (e.g.,an amplification product produced by the amplification methods describedherein). In embodiments, the sequencing step may be accomplished by asequencing-by-synthesis (SBS) process. In embodiments, sequencingcomprises a sequencing by synthesis process, where individualnucleotides are identified iteratively, as they are polymerized to forma growing complementary strand. In embodiments, nucleotides added to agrowing complementary strand include both a label and a reversible chainterminator that prevents further extension, such that the nucleotide maybe identified by the label before removing the terminator to add andidentify a further nucleotide. Such reversible chain terminators includeremovable 3′ blocking groups, for example as described in U.S. Pat. Nos.10,738,072, 7,541,444 and 7,057,026. Once such a modified nucleotide hasbeen incorporated into the growing polynucleotide chain complementary tothe region of the template being sequenced, there is no free 3′-OH groupavailable to direct further sequence extension and therefore thepolymerase cannot add further nucleotides. Once the identity of the baseincorporated into the growing chain has been determined, the 3′ blockmay be removed to allow addition of the next successive nucleotide. Byordering the products derived using these modified nucleotides it ispossible to deduce the DNA sequence of the DNA template. Non-limitingexamples of suitable labels are described in U.S. Pat. Nos. 8,178,360,5,188,934 (4,7-dichlorofluorscein dyes); U.S. Pat. No. 5,366,860(spectrally resolvable rhodamine dyes); U.S. Pat. No. 5,847,162(4,7-dichlororhodamine dyes); U.S. Pat. No. 4,318,846 (ether-substitutedfluorescein dyes); U.S. Pat. No. 5,800,996 (energy transfer dyes); U.S.Pat. No. 5,066,580 (xanthene dyes): U.S. Pat. No. 5,688,648 (energytransfer dyes); and the like.

Sequencing includes, for example, detecting a sequence of signals. Inembodiments, sequencing includes detecting a sequence of signals andgenerating one or more sequencing reads. Examples of sequencing include,but are not limited to, sequencing by synthesis (SBS) processes in whichreversibly terminated nucleotides carrying fluorescent dyes areincorporated into a growing strand, complementary to the target strandbeing sequenced. In embodiments, the nucleotides are labeled with up tofour unique fluorescent dyes. In embodiments, the nucleotides arelabeled with at least two unique fluorescent dyes. In embodiments, thereadout is accomplished by epifluorescence imaging. A variety ofsequencing chemistries are available, non-limiting examples of which aredescribed herein.

Flow cells provide a convenient format for housing an array of clustersproduced by the methods described herein, in particular when subjectedto an SBS or other detection technique that involves repeated deliveryof reagents in cycles. For example, to initiate a first SBS cycle, oneor more labeled nucleotides and a DNA polymerase in a buffer, can beflowed into/through a flow cell that houses an array of clusters. Theclusters of an array where primer extension causes a labeled nucleotideto be incorporated can then be detected. Optionally, the nucleotides canfurther include a reversible termination moiety that temporarily haltsfurther primer extension once a nucleotide has been added to a primer.For example, a nucleotide analog having a reversible terminator moietycan be added to a primer such that subsequent extension cannot occuruntil a deblocking agent (e.g., a reducing agent) is delivered to removethe moiety. Thus, for embodiments that use reversible termination, adeblocking reagent (e.g., a reducing agent) can be delivered to the flowcell (before, during, or after detection occurs). Washes can be carriedout between the various delivery steps as needed. The cycle can then berepeated N times to extend the primer by N nucleotides, therebydetecting a sequence of length N. Example SBS procedures, fluidicsystems and detection platforms that can be readily adapted for use withan array produced by the methods of the present disclosure aredescribed, for example, in Bentley et al., Nature 456:53-59 (2008), USPatent Publication 2018/0274024, WO 2017/205336, US Patent Publication2018/0258472, each of which are incorporated herein in their entiretyfor all purposes.

Use of the sequencing method outlined above is a non-limiting example,as essentially any sequencing methodology which relies on successiveincorporation of nucleotides into a polynucleotide chain can be used.Suitable alternative techniques include, for example, pyrosequencingmethods, FISSEQ (fluorescent in situ sequencing), MPSS (massivelyparallel signature sequencing), or sequencing by ligation-based methods.

In embodiments, each particle core includes a silica, magnetic, orparamagnetic material, such as in the form of a bead or particle. Forexample, the particle shell layers may be formed around andencapsulating a supporting bead, for example, a silica, magnetic, orparamagnetic bead.

In embodiments, the particle is a functionalized particle including aparticle core and a particle shell, wherein said particle shell includesthe plurality of bioconjugate reactive moieties, the plurality ofoligonucleotide moieties, or a combination thereof, wherein each of thebioconjugate reactive moieties and each of the oligonucleotide moietiescomprise a linker binding said bioconjugate reactive moieties andoligonucleotide to the particle core. In embodiments, the functionalizedparticle is a silica particle.

In embodiments, the particle is a functionalized particle including aparticle core and a plurality of particle polymers. In embodiments, eachparticle polymer includes a plurality of bioconjugate reactive moieties,a plurality of oligonucleotide moieties, or a combination thereof. Inembodiments, the functionalized particle is a silica particle. Inembodiments of the methods provided herein, the particle is a polymericparticle.

In embodiments, arraying the particles includes contacting the surfacewith a first solution comprising the plurality of particles in ananti-solvent. In embodiments, the particle includes acrylamide and theanti-solvent is an aqueous ethanol solution. In embodiments, theparticle includes sulfobetaine acrylate (SBA), carboxybetaine acrylate(CBA), phosphorylcholine acrylate (PCA), sulfobetaine methacrylate(SBMA), carboxybetaine methacrylate (CBMA), or phosphorylcholinemethacrylate (PCMA) and the anti-solvent is an aqueous acetone solution.In embodiments, arraying the particles further includes removing thefirst solution and contacting the surface with a second solution,wherein the second solution is an aqueous solution capable of expandingthe volume of the particle. In embodiments, the second solution includeswater. In embodiments of the methods provided herein, the secondsolution expands the volume of the particles by up to 10%, up to 20%, upto 30%, up to 40%, up to 50%, up to 60%, up to 70%, up to 80%, up to100%, up to 150%, up to 200%, or more relative to particle in ananti-solvent. In embodiments of the methods provided herein, the solventexpands the volume of the particles by 10-200%, 30-150%, or 50-100%,relative to particle in an anti-solvent. In embodiments of the methodsprovided herein, the presence of the solvent expands the volume of theparticles by up to 90% relative to particle in an anti-solvent.

In embodiments, the polymers of the present disclosure (e.g., polymerparticles) swell with a solvent in which they are suspended, and therefractive index of the suspension is about the same as the solvent. Inembodiments of the methods provided herein, the polymer particle or theshell polymer, have a refractive index of about 1.2-1.6, 1.25-1.5, or1.3-1.4 when hydrated. In embodiments, the polymer particle or the shellpolymer have a refractive index of about 1.3 when hydrated.

In embodiments, the polymers of the present disclosure (e.g., polymericparticles) swell with a solvent in which they are suspended, and therefractive index of the suspension is about the same as the solvent. Inembodiments of the methods provided herein, the polymeric particle orthe shell polymer, have a refractive index of about 1.2-1.6, 1.25-1.5,or 1.3-1.4 when hydrated. In embodiments, the polymeric particle or theshell polymer have a refractive index of about 1.3 when hydrated.

In an aspect, provided herein are methods of sequencing targetpolynucleotides, the methods including contacting a polymer particlewith a sample that includes target polynucleotides, amplifying thetarget polynucleotides to produce discrete amplicon clusters, andsequencing the amplicon clusters. In embodiments, the polymer particleincludes a polymer covalently attached to polynucleotide primers. Inembodiments, amplifying the target includes extension of primers alongthe target polynucleotides within the polymer particle. In embodiments,each amplicon cluster originates from amplification of a single targetpolynucleotide. In embodiments, sequencing comprises detecting sequencesof signals within the polymer particle.

In an aspect, provided herein are methods of sequencing targetpolynucleotides, the methods including contacting a polymeric particlewith a sample that includes target polynucleotides, amplifying thetarget polynucleotides to produce discrete amplicon clusters, andsequencing the amplicon clusters. In embodiments, the polymeric particleincludes a polymer covalently attached to polynucleotide primers. Inembodiments, amplifying the target includes extension of primers alongthe target polynucleotides within the polymeric particle. Inembodiments, each amplicon cluster originates from amplification of asingle target polynucleotide. In embodiments, sequencing comprisesdetecting sequences of signals within the polymeric particle.

In embodiments of the methods provided herein, the targetpolynucleotides are at a concentration in the sample selected to produceamplicon clusters having a desired density. For example, theconcentration of target polynucleotides is selected based on acalculation of (a) the average size of a cluster of amplicons that willresult from amplification under selected conditions (e.g. a selectedduration and number of extension steps), and (b) a desired separationbetween adjacent amplicon clusters in the array.

In embodiments of the methods provided herein, the amplicon clustershave a mean or median separation from one another of about 0.5-5 μm. Inembodiments, the mean or median separation is about 0.1-10 microns,0.25-5 microns, 0.5-2 microns, 1 micron, or a number or a range betweenany two of these values. In embodiments, the mean or median separationis about or at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9,1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3,2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7,3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0 μm or anumber or a range between any two of these values. The mean or medianseparation may be measured center-to-center (i.e., the center of oneamplicon cluster to the center of a second amplicon cluster). Inembodiments of the methods provided herein, the amplicon clusters have amean or median separation (measured center-to-center) from one anotherof about 0.5-5 μm. The mean or median separation may be measurededge-to-edge (i.e., the edge of one amplicon cluster to the edge of asecond amplicon cluster). In embodiments of the methods provided herein,the amplicon clusters have a mean or median separation (measurededge-to-edge) from one another of about 0.2-5 μm.

Neighboring features of an array can be discrete one from the other inthat they do not overlap. Accordingly, the features can be adjacent toeach other or separated by a gap (e.g., an interstitial space). Inembodiments where features are spaced apart, neighboring sites can beseparated, for example, by a distance of less than 10 μm, 5 μm, 1 μm,0.9 μm, 0.8 μm, 0.7 μm, 0.6 μm, 0.5 μm, or less. The layout of featureson an array can also be understood in terms of center-to-centerdistances between neighboring features. An array useful in the inventioncan have neighboring features with center-to-center spacing of less thanabout 10 μm, 5 μm, 1 μm, 0.9 μm, 0.8 μm, 0.7 μm, 0.6 μm, 0.5 μm or less.Furthermore, it will be understood that the distance values describedabove and elsewhere herein can represent an average distance betweenneighboring features of an array. As such, not all neighboring featuresneed to fall in the specified range unless specifically indicated to thecontrary, for example, by a specific statement that the distanceconstitutes a threshold distance between all neighboring features of anarray.

In embodiments, each feature generated on the surface of an array can beof similar or smaller size than the area of the surface occupied by theparticle from which the feature was produced, and all the features willtypically be of similar size and intensity to each other. The uniformsize, uniform intensity, and lack of overlap provides a convenientdensity of features per unit area. Detection of tightly packednon-overlapping arrays with features of uniform size and intensity aretypically easier to analyze than images where a subset of the featuresoverlap with each other.

In embodiments of the methods provided herein, the amplicon clustershave a mean or median diameter of about 100-2000 nm, or about 200-1000nm. In embodiments, the mean or median diameter is about 100-3000nanometers, about 500-2500 nanometers, about 1000-2000 nanometers, or anumber or a range between any two of these values. In embodiments, themean or median diameter is about or at most about 100, 200, 300, 400,500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700,1800, 1900, 2000 nanometers or a number or a range between any two ofthese values.

In embodiments, the arrays include about 10,000,000 features/cm² toabout 5,000,000,000 features/cm². In embodiments, the arrays includeabout 100,000,000 features/cm² to about 1,000,000,000 features/cm². Inembodiments, the arrays include about 100,000 features/cm² to about100,000,000 features/cm². In embodiments, the arrays include about orabout 10,000,000 features/cm² to about 50,000,000 features/cm².

In embodiments of the methods provided herein, the contacting step isperformed under non-hybridizing conditions. Examples of non-hybridizingconditions are described above, and include but are not limited to lowsalt, high temperature, or presence of additives such as formamide.

EXAMPLES Example 1. Superior Coverage of Nucleic Acid Templates withinNanoarrays

Patterned arrays are an important tool in biomedical research, providinga two-dimensional platform that arranges biological samples and enableshigh-throughput analyses. Delivering breakthroughs in proteomics,multiplexed immunoassays, and complex genomic analyses, microarrays canbe designed to host thousands, or even ten-thousands, of features thatcan be subjected to simultaneous reaction conditions. Microarrays aretypically fabricated by spotting, imprinting, or directly synthesizingbiomolecules on solid supports such as glasses, silicon wafers, andother functionalized substrates. In general terms, a target of interest(e.g., a protein or gene sequence) is immobilized as discrete features,or spots, on a substrate. Each feature may contain one to thousands ofidentical targets if subjected to an amplification technique. Asuccessful detection event occurs when a labeled probe is brought intocontact with the array, and if the probe interacts with the target, anincrease of fluorescence intensity over a background level is produced,which can be measured using an appropriate detector.

Array techniques that rely on the random distribution of featurestypically suffer from a low ratio of incorporation event/pixel, due to ahigh number of dark pixels with no features (for example, if the densityof features is too diffuse), or a high number of pixels that carrymultiple overlapping features of different sequence (if the density offeatures is too concentrated) or both (due to the random nature offeature placement). An ideal and more efficient use of the imagingpixels occurs when the features on the surface are tightly packed,non-overlapping, and of similar size and intensity to each other. Thepresent invention provides compositions and methods of manufacturingarrays of features that avoid low ratios of bases/pixels associated withtypical commercial array fabrication methods, while exploitingadvantages of random feature fabrication. Maximizing the number oftarget polynucleotides per surface area will enable scientists toanalyze a complex genome on one small glass chip, about 1 cm² in size.

Although the microarray has become a mainstay for parallel screening ofseveral nucleic acids and proteins, it has several disadvantages. Forexample, microarray applications require large sample volumes and longincubation times because of the larger spot size (e.g., 1-150 μm).Bead-based microarrays were developed by David Walt at Tufts Universityand subsequently commercialized by BeadChip products (Walt, D. R.Science 2000, 287(5452), 451-452) and others (Brenner et al. Nat.Biotechnol. 2000, 18, 630-634), however, the large diameter of the beads(e.g., 3 μm to 40 μm) limit the theoretical maximum density andpractical use of the underlying array. A significant reduction inparticle size is necessary to achieve higher throughput, less reagentconsumption, and faster data acquisitions. For context, the averagediameter of a grain of sand is 60 to 2,000 μm (“Relationship ofTransported Particle Size to Water Velocity.” 1994 Earth ScienceReference Tables. Albany, N.Y.: University of the State of New York,1994), and manipulating nanoparticles is challenging. Particlessuspended in liquids are prone to form aggregates, and given the uniqueproperties pertinent to nanoparticles, such as shape, size, surfacecharacteristics, composition, and electronic structures, nanoparticleaggregation is more problematic than their bulk counterparts (i.e.,microparticles). Further reducing the bead size to submicron dimensions,while not aggregating and retaining the necessary functional propertiesto form highly dense arrays, withstand repeated cycles of complexbiochemical processes that result in signal generation and detection,has until now, remained elusive.

In embodiments, the array and associated particles as described hereinaddress the problem of achieving a high array density with complete tonear-complete loading of particles into wells of an array, enablinggreater coverage of monoclonal templates per cm². Highly efficientloading is achieved with a pattern of high-density particle-occupancysites separated by a non-binding surface (e.g., interstitial space),wherein the particle type and size, preparation methods, and areas ofdiscrete spaced apart regions are selected so that substantially allsuch regions contain at most only one single particle. An array thatachieves high coverage of monoclonal templates is advantageous fordetection and data analysis of signals collected from the arrays duringsequencing analysis. Tuning the particle size allows for additionalcontrol of the quantity of particles per well (e.g., 2 or more particlesper well).

Polynucleotides on particles of the invention herein, confined to therestricted area of discrete, punctate clusters provide a moreconcentrated or intense signal, particularly when fluorescent probes areused in analytical operations, thereby providing higher signal-to-noisevalues and greater confidence in detection. By generating punctateclusters in an array that will provide a signal, data collection issimplified and less sophisticated image analysis systems are needed todetect fewer pixels compared to traditional systems. As described supra,a benefit provided by embodiments of the array may include an increasedsignal intensity during sequencing-by-synthesis. The increase in signalintensity may reduce an error rate by reducing the number of clusters,for example, that emit a low intensity of light. Maintaining theinterstitial spaces of the array free from non-specifically boundoligonucleotides, and subsequently amplified clusters, would helpquarantine signal production to regions that have a potential broaderdynamic range of signal emittance.

A benefit of the invention described herein may include a decrease insignal to noise ratio that enables faster scan speeds and reducesoverall time for conducting a protocol. For instance, with respect tosequencing-by-synthesis technology, faster scan speeds on sequencinginstruments are desired, but faster scan speeds result in fewer photonsbeing collected per cluster on the imaging camera. With fewer photonscaptured, the signal to noise ratio typically decreases and it becomesmore difficult to confidently assign a base. Furthermore, on somesequencing instruments, low NA optics result in signals that areinherently larger and dimmer, potentially yielding higher error rates.Embodiments set forth herein may increase the number of photons that arecaptured by eliminating regions of non-specific binding. The increase insignal intensity may improve overall sequencing performance by reducingsequencing error arising from low intensity clusters (e.g., those foundin interstitial spaces) and cluster dropouts during long sequencingruns.

The array of the invention described herein may include particles ofvarious compositions. In some embodiments, the particles may includesolid core particles (e.g., a core made of glass, ceramic, metal,silica, magnetic material, or a paramagnetic material) with a pluralityof particle polymers (e.g., a particle polymer moieties includingpolyacrylamide (Aam), poly-N-isopropylacrylamide, polyethylene glycolacrylate, methacrylate, acrylamide/N,N′-bis(acryloyl)cystamine (BACy),PEG/polypropylene oxide (PPO), polyacrylic acid, poly(hydroxyethylmethacrylate) (PHEMA), poly(methyl methacrylate) (PMMA),poly(N-isopropylacrylamide) (PNIPAAm), poly(lactic acid) (PLA),poly(lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL),poly(vinylsulfonic acid) (PVSA), poly(L-aspartic acid), poly(L-glutamicacid), glicydyl methacrylate (GMA), hydroxyethylmethacrylate (HEMA),hydroxyethylacrylate (HEA), hydroxypropylmethacrylate (HPMA),polyethylene glycol methacrylate (PEGMA), polyethylene glycol acrylate(PEGA), isocyanatoethyl methacrylate (IEM), or a copolymer thereof). Inembodiments, the solid core particle is a silica particle. Inembodiments, the particle polymer includes PEGMA and GMA azidecopolymers. In other embodiments, the array includes particles made upentirely of a polymeric scaffold (e.g., including a copolymer describedsupra). The polymeric particle is permeable to sequencing reactionmixtures and amplification reaction mixtures, including reagents,oligonucleotides, and polymerases. The polymer compositions providedherein prevent nanoparticle aggregation.

One feature of the invention described herein includes an array withwells containing particles dispersed into the array, wherein theparticles are in the sub-micron diameter (i.e., the diameter of theparticle is less than or equal to about 1 μm) range. As shown in FIG. 8, pure particles below 1000 nm in diameter may be utilized incombination with an array of the invention. Particles with smallerdiameters, for example, diameters of about 500 nm to 400 nm or smaller,may be implemented to increase the array loading efficiency and highcoverage. We have validated the ability to deposit such sub-micronparticles in an array with high coverage (see, FIG. 4 , for example). Inembodiments, the particle has a diameter of about 400 nm. Inembodiments, the particle has a diameter of about 450 nm. Inembodiments, the thickness of the surrounding particle polymers (alsoreferred to herein as the particle shell) is about 50 to about 200 nm(see FIG. 10A). In embodiments, the thickness of the copolymeric shellis about 50 nm. In embodiments, the thickness of the copolymeric shellis about 100 nm. In embodiments, the thickness of the copolymeric shellis about 150 nm. In embodiments, the thickness of the copolymeric shellis about 200 nm.

Particles of the invention may be loaded into wells through severalmethods known in the art. For example, particles loading may simply begravity driven. Gravity driven loading may also be accelerated bysubsequently spinning down the array in a centrifuge, or with an orbitalmixer to increase the particle settling rate. Such combinations areoptimized so that no more than one particle is loaded into a given well,while achieve near complete coverage of the array with high uniformity.Additional particle loading techniques may involve agitating (e.g.,vortexing), capillary assisted wetting, and/or centrifugation. In otherembodiments, sonication and/or physical wiping with a flat tool may beused as a post-loading cleaning technique to reduce doubly-loaded wellsand clear interstitial regions of particles. Post-cleaning may alsosimply consist of rinsing with a solvent, shaking, sonicating, wiping,or a combination thereof to remove non-specifically bound particles.

An alternative approach to arraying particles into wells is illustratedin FIG. 3 . In this approach, oligonucleotide primer-coated particlesare introduced in an anti-solvent (e.g., ethanol). In the presence ofanti-solvent the volume of the primer-coated particles is reducedrelative to the volume of the primer-coated particles in solvent.Subsequently, the reaction conditions are changed such that a solvent(e.g., water) is present, which swells the particles, causing them toremain trapped in the wells.

The particles are decorated with bioconjugate reactive moieties (e.g., aplurality of bioconjugate reactive moieties, depicted as small dots inFIG. 2A), such that either before, or after, loading the particles intoa well, one or more oligonucleotide moieties may be bound to theparticles, as depicted in FIG. 2B and imaged in FIGS. 5A-5B. In someembodiments, the oligonucleotides moiety is about 5 to about 45nucleotides in length and is capable of hybridizing to a library nucleicacid molecule. In embodiments, the oligonucleotide moiety is capable ofhybridizing to a complementary sequence of a template nucleic acid.

In order to obtain high-density particle loading with minimalbackground, the solid support of the array may be coated with apassivating polymer (e.g., a copolymer such as a silane functionalizedpolyethylene glycol (Si-PEG) copolymer or a silane functionalizedpoly(acrylamide) (Si-Pam)). The passivating polymer may be hydrophilicor hydrophobic (e.g., polyfluorinated polymer) and may be a comb polymeror brush polymer that is useful at preventing non-specific binding ofadditional agents to the array (e.g., oligonucleotides in solution).Illustrations of the different well shapes of the array are depicted inFIGS. 1A-1C. Using known nanolithographic fabrication techniques, aglass substrate may be etched such that the well is anisotropic (FIG.1A), partially anisotropic (FIG. 1B), or isotropic (FIG. 1C). The arraymay include a photoresist (e.g., a fluorinated polymer later) prior toreceiving an additional polymer coating (e.g., a poloxamer oralkoxysilyl polymer). The photoresist may be removed prior to theaddition of the additional polymer using known techniques in the art(e.g., solvent removal). In embodiments, the additional polymer coatingreduces the non-specific binding of oligonucleotide moieties. Inembodiments, the particles are physiosorbed to the surface of the wells.Surprisingly, no covalent linkage between the particle and the array,nor hybridization of particles bearing an oligonucleotide sequence thatis complementary to an immobilized primer on the array, is needed toretain the particles in the well. The interaction between the particleand the passivating polymer is sufficient to retain the particles in thewell during amplification and sequencing.

The array may include a nanoimprint resist (e.g., organically-modifiedceramic polymers, such as OrmoComp® from micro resist technology GmbH),which contains the plurality of wells (e.g., see FIG. 1D).Organically-modified ceramics are hybrid polymers with inorganic andorganic moieties linked by stable covalent bonds and based onorganically modified alkoxysilanes, functionalized organic polymers orboth. See K. H. Haas, H. Wolter, “Synthesis, properties and applicationsof inorganic-organic copolymers (ORMOCER®s),” Current Opinions in SolidState and Materials Science, vol. 4, pp. 571-580, 1999, which isincorporated herein by reference. In nanolithography technologies,organically-modified ceramic polymers behave similarly to negative-tonephotoresists, such as SU-8, and provide glass-like material propertiesafter UV curing. Typical organically-modified ceramic polymers includeoxides (e.g., SiO₂, ZrO, MgO, Al₂O₃, TiO₂ or Ta₂O₅), silicon oxide(—Si—O—) groups, polymerizable monomers (e.g., acryl or methacrylatemonomers), and one or more alkyl moieties. In embodiments, theorganically-modified ceramic polymer includes alkoxysilane and/orpolymerized units of alkoxysilyl monomers. In embodiments, theorganically-modified ceramic polymer includes polymerized monomers of

In embodiments, the organically-modified ceramic polymer is stable(i.e., does not measurably degrade) up to about 300° C.

For example, by coating the array with a brush hydrophilic polymer(e.g., a random copolymer of p[PEGMA-co-TESPM]), significantly lessdetectable oligonucleotides in the interstitial regions followingcluster amplification of hybridized template nucleic acids, compared toa control particle-loaded array lacking a hydrophilic polymer coating(see, FIGS. 6A-6B) is observed. The array may also be coated with apolymer (e.g., a random copolymer of p[PEGMA-co-HEMA-co-TMSPM] orp[PEGMA-co-HEMA] to provide surfaces with a visible nanopattern (seeFIGS. 9A-9C). The arrayed particles may then be subjected to seedingwith template polynucleotides. Additionally, following seeding, thearrayed particles may then be subjected to amplification and sequencing(e.g., sequencing-by-synthesis) to determine the identity of thepolynucleotide template(s) attached to each discrete particle in thearray.

Example 2. Synthetic Materials and Methods

Synthesis of polymeric particles useful for the methods and compositionsdescribed herein may be synthesized as follows. Synthesis of polymericcores were performed by dispersion polymerization in water/alcoholmixtures in which monomer(s) are soluble. AAm and azide functionalizedmonomers were added to the reactor along with a stabilizer, FRPinitiator and solvent and mixed. The reaction vessel was sealed andbubbled with inert gas in the ice bath. A crosslinker (e.g., bis-AAm)was dissolved in water/alcohol mixture in another tube and bubbled withinert gas, simultaneously. After around 30 min, main reaction vessel washeated to above 50° C. and mixed via stirrer bar in the constant rate.The crosslinker solution was added slowly. Other functional monomer(s)can be added during or after the crosslinker addition to the mainreactor. For example, 25 mg Ammonium persulfate (APS), 0.5 g AAm, 130 mgof GMA-N₃, 1 g of PVP (average mol wt 40,000), 16 g ethanol and 4 gwater added to the first reaction vessel along with magnetic stirrerbar. The reaction vessel was sealed and purged with inert gas for atleast 30 min and cooled. In another container, 8 g of ethanol, 2 g waterand 24 mg N,N′-Methylenebis(acrylamide) mixed and bubbled with inert gas(solution 2). After around 30 min, main reaction vessel was heated toabove 50° C. and mixed via stirrer bar in the constant rate. Thecrosslinker solution was added slowly, followed by addition of 11 ul ofglycidyl methacrylate (GMA). The particles may further be modified byconverting the epoxy to azide using known techniques in the art (e.g.,aqueous sodium azide). In another example, 25 mg Ammonium persulfate(APS), 0.5 g AAm, 75 mg of HEMA-N3, 0.4 g of PVP (average mol wt40,000), 17 g ethanol and 3 g water were added to the reactor(solution 1) along with a magnetic stirrer bar. The reactor was sealedand purged with an inert gas for at least 30 minutes, in an ice bath. Toform the crosslinker solution, in another container, 8.5 g of ethanol,1.5 g water and 24 mg N,N′-Methylenebis(acrylamide) was mixed andbubbled with inert gas (solution 2). The reactor is immersed in an oilbath (60° C.) to start the reaction while mixed at a 120 rpm stirringrate. After 20 minutes, solution 2 is added to the reactor with asyringe pump (at a rate of 6 ml/h). At the end of crosslinker solutionaddition, 11 ul of glycidyl methacrylate (GMA) was added to remainingsolution. It was bubbled and then continued to be added to the mainreactor to achieve epoxy decorated functionalized particles.

Surface Initiated ATRP Particle Polymerization General Methods. Thesubstrate particle (e.g., silica particle or metal particle) containinga polymerization initiator is immersed in polymerization reactionmixture as described above. This mixture depends on the type ofmonomers, and can include (i) solvent(s), monomer(s), initiator, andligand or (ii) solvent(s), monomer(s) and initiator. Monomer ratios wereadjusted to create polymers brushed with different spacers betweenneighboring side chains (ng) (i.e., determining the ratio of monomerswith functional groups, e.g., azide or alkyne moieties, to monomers withnon-functional groups, e.g., PEG). In embodiments, the ratio offunctional groups to non-functional groups is 1:1. In embodiments, theratio of functional groups to non-functional groups is 1:2. Inembodiments, the ratio of functional groups to non-functional groups is1:3. In embodiments, the ratio of functional groups to non-functionalgroups is 1:4. In embodiments, the ratio of functional groups tonon-functional groups is 1:5. In embodiments, the ratio of functionalgroups to non-functional groups is 1:6. In embodiments, the ratio offunctional groups to non-functional groups is 1:7. In embodiments, theratio of functional groups to non-functional groups is 1:8. Modulatingthe ng (i.e., the ratio of functional groups to non-functional groups ofthe copolymer allows for control of the density of oligonucleotides.

In embodiments, the polymer does not include N-(5-bromoacetamidylpentyl)acrylamide (BRAPA). The polymers and copolymers described herein haveincreased solubility in water and have a greater density of functionalgroups (e.g., a ratio of non-reactive groups such as PEG to reactivegroups such as azide, is 3:1) as compared to acrylamide-based copolymers(e.g., N-(5-bromoacetamidylpentyl) acrylamide (BRAPA)). Typicalacrylamide based polymers and copolymers need to increase the ratio offunctional groups to non-functional groups (NR:R) around 8:1 to 20:1 tomaintain solubility, which ultimately limits the density ofoligonucleotides.

Synthesis of GMA-azide. Starting with sodium azide (NaN₃), it wasdissolved in deionized water and the pH was reduced by dropwise additionof HCl to achieve a pH of 5.0. Approximately 30 mL of glycidylmethacrylate (GMA) was added to the sodium azide mixture via a syringeand stirred overnight to generate a mixture GMA-azides having the

major and minor structures 3-azido-2-hydroxypropyl methacrylate and

2-azido-3-hydroxypropyl methacrylate, confirmed via NMR. The GMA-azidewas then purified.

Synthesis of 2-(((2-azidoethoxy)carbonyl)amino)ethyl methacrylate.Starting with sodium azide (NaN₃), it was dissolved in deionized waterand 30 mL of 2-bromoethanol was added via syringe. The reaction wasmaintained at 55° C. and stirred overnight. Approximately 25 g of2-Isocyanatoethyl methacrylate (IEM) was added to the azido-ethanol(N₃EtOH) mixture and stirred at room temperature for 2-3 hours. Asolution of dibutyltin dilaurate (DBTDL) and dichloromethane (DCM) wastransferred to the IEM mixture and stirred overnight at room temperatureto generate IEM-azide. The IEM-azide was then purified and confirmed viaNMR to have the structure

2-(((2-azidoethoxy)carbonyl)amino)ethyl methacrylate.

Example 3. Preparation of Silica Particles

Functionalization of Silica Particles. Silica and deionized (DI) waterare mixed in a vessel and sonicated at room temperature. Once theparticles are dispersed, tetrabutylammonium hydroxide (55% TBAH insolution) was mixed with the solution and placed into a closed system,stirred under ventilation for 24 hours at 60° C. The solution iscentrifuged, and the liquid is discarded. The remaining particles aremixed with DI water then sonicated at room temperature. The particlesare washed with DI water until the pH of the solution is 7. Theparticles are centrifuged, and the supernatant is discarded. Apolymerization initiator compound (i.e., an ATRP initiator),(3-trimethoxysilyl)propyl 2-bromo-2-methylpropionate is added dropwiseto the particles in solution and mixed for 24 hours at room temperature.The particles are then washed and redispersed by sonication to giveparticles modified with ATRP polymerization initiator.

Polymerization of Silica Particles. Through a column of basic alumina,PEGMA₅₀₀, Mn=500 was passed through to remove BHT inhibitor. Thepurified PEGMA₅₀₀ was added to ATRP-initiator functionalized silicaparticles in ethanol in a flask and sonicated until the particles aredispersed. GMA-Azide, 2-hydroxyethyl 2-bromoisobutyrate (OH-EBiB),CuBr₂, 1,1,4,7,10,10-Hexamethyltriethylenetetramine (HMTETA) was thenadded to the reaction flask. Copper(I) bromide (CuBr) is added to thereaction flask and the flask was heated to 60° C. The reaction mixturewas rinsed, centrifuged, and the supernatant is removed. Fresh ethanolwas added to the particles, and this solution is vortex and sonicatedseveral times. The supernatant was removed to provide polymerized silicaparticles that were examined under microscope.

Addition of Oligonucleotide Primers to Polymerized Silica Particles. Ina reactor containing ethanol/buffer solution oligonucleotide primerswere added. To this same reactor, polymerized silica particles are addedand the mixture was sonicated and stirred overnight. The resultingpolymerized silica particles with oligonucleotide primers are spun downand washed before being dispersed in an ethanol/salt solution. Followingsonication, the particles were stored in solution.

Example 4. Preparation of Polymerized Surface Slides

Into an air free reaction vessel, azobisisobutyronitrile (AIBN) is addedfollowed by 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid (CTA),purified PEGMA, Mn=500 and purified TMSPM. Anhydrous toluene andanhydrous acetone are added to the reaction vessel, and everything ismixed under N₂ for 1 hr in ice then stirred at an increased temperature(e.g., 50° C. to 70° C.) overnight.

A solution of polymer in toluene is poured into a glass reaction vesseland glass slides (e.g., patterned slides) are then added to this vessel.The reaction vessel is placed in a desiccator and sealed for 15-18 hrs.The slides were placed into another container containing toluene,sonicated, and then placed into a container containing ethanol andsonicated again.

Example 5. Particle Loading

The following are examples of general particle loading techniques whichmay be applied in carrying out the invention. To a patterned glass slidecontaining a photoresist with wells spaced 1.0 μm apart(center-to-center), a copolymer of PEGMA-co-TMSPM is applied to thesurface. The ratio of alkoxysilyl groups to PEG groups was 1:8.Approximately 4 mL of a colloidal solution containing approximately 10¹⁰silica core particles (400 nm diameter) having a PEGMA-co-GMA azideshell were incubated with the array and allowed to settle into thewells. The ratio of azide functional groups to PEG functional groups was1:4. The colloidal solution included ethanol, isopropyl alcohol, andwater. The incubated array may be subjected to centrifugation (e.g.,2000 RPM for 12 minutes) or vortexed (e.g., 300 RPM for 12 minutes) toaccelerate particle loading. Following loading, the array was washed 3×with water or ethanol and sonicated for 3 minutes. An additional washstep is performed with an ethanol, isopropyl alcohol, and watersolution. The particle loaded array is allowed to dry or may be wipeddown to accelerate surface drying. The array may be stored in an aqueoussolution until ready to use.

To a patterned glass slide containing a photoresist with wells spaced1.4 μm apart (center-to-center), a copolymer of PEGMA-co-TMSPM isapplied to the surface. The ratio of alkoxysilyl groups to PEG groupswas 1:8. Approximately 4 mL of a colloidal solution containingapproximately 10¹⁰ silica core particles (500 nm diameter) having aPEGMA-co-GMA azide shell were incubated with the array and allowed tosettle into the wells. The ratio of azide functional groups to PEGfunctional groups was 1:4. The colloidal solution included ethanol,isopropyl alcohol, and water. The incubated array was subjected tocentrifugation (e.g., 2000 RPM for 12 minutes) to accelerate particleloading. Following loading, the array was washed 3× with water orethanol and sonicated for 3 minutes and allowed to dry. An additionalwash step is performed with an ethanol, isopropyl alcohol, and watersolution. The particle loaded array is allowed to dry or may be wipeddown to accelerate surface drying. The array may be stored in an aqueoussolution until ready to use.

Quality control experiments were performed on the particle loadedarrays. Approximately 95% of the available wells had a single particle.Following primer deposition, i.e., the oligonucleotide moietiescovalently attached to the reactive moieties on the particles, aFAM-labeled complement was incubated in the array and imaged. As shownin FIGS. 5A-5B, the fluorescent intensity of the discrete particles isobserved and is in contrast to the reference fiducial (i.e., the centercircle in FIGS. 5A-5B) and the interstitial regions which do not containany FAM-labeled complements, and thus do not include any oligonucleotideprimers.

The particles were physiosorbed in the wells. Surprisingly, no covalentlinkage nor tethering to immobilized oligonucleotides between theparticle to the well was used. Efforts to remove the particles fromwells included inverting the array, wiping, rinsing, and sonicatingovernight in ethanol and toluene. Quality control experiments comparingthe FAM-labeled probe intensity before and after sonicating wereindistinguishable, indicating the particles remain in the wells, despitethe aggressive treatments.

Polymerized silica particles are added to a mixture of ethanol,isopropyl alcohol and water and sonicated. Into this solution was addeda polymerized surface slide (i.e., a passivated slide). The slide andparticle solution are shaken and incubated for 1 to 8 hours. Followingthis incubation period, the slides are shaken in an ethanol solutionseveral times. To check the quantity of synthesized particles depositedonto the polymerized surface slides, phase contrast microscopy wasperformed on the slides.

Polymerized silica particles with oligonucleotide primers in loadingsolution (TE buffer with NaCl which may optionally contain ethanol) issonicated. Following sonication, the particles in solution was added toa tray containing patterned glass slide(s) that contain a resist, forexample SU-8 and/or Ormocomp®, and shaken for 10 min. Following shaking,the tray is placed at 4° C. The slides are dried, washed and driedagain. The slides were examined under microscope to check the quantityof polymerized silica particles with oligonucleotide primers particlesdeposited on the slides.

EMBODIMENTS

The present disclosure provides the following additional illustrativeembodiments.

Embodiment P-1. An array, comprising: a solid support comprising asurface, the surface comprising a plurality of wells separated from eachother by interstitial regions on the surface, wherein the surfacecomprises a polymer layer and is substantially free of oligonucleotidemoieties, wherein one or more wells contains a particle, wherein theparticle comprises a plurality of bioconjugate reactive moieties, aplurality of oligonucleotide moieties, or a combination thereof.

Embodiment P-2. An array, comprising: a solid support comprising asurface, the surface comprising a plurality of wells separated from eachother by interstitial regions on the surface, wherein one or more wellscontains a particle, wherein the particle comprises a plurality ofbioconjugate reactive moieties, a plurality of oligonucleotide moieties,or a combination thereof; and the average longest dimension of theparticle is from about 100 nm to about 1000 nm.

Embodiment P-3. The array of Embodiment P-1 or P-2, wherein there is atleast one particle per well.

Embodiment P-4. The array of Embodiment P-1 or P-2, wherein there is oneparticle per well.

Embodiment P-5. The array of any one of Embodiments P-1 to P-4, whereinthe particle is a functionalized particle comprising a particle core anda particle shell, wherein said particle shell comprises the plurality ofbioconjugate reactive moieties, the plurality of oligonucleotidemoieties, or a combination thereof, wherein each of said bioconjugatereactive moieties and each of said oligonucleotide moieties comprise alinker binding said bioconjugate reactive moieties and oligonucleotideto said particle core.

Embodiment P-6. The array of Embodiment P-5, wherein the particle corecomprises glass, ceramic, metal, silica, magnetic material, or aparamagnetic material.

Embodiment P-7. The array of Embodiment P-5, wherein the particle shellcomprises polymerized units of polyacrylamide (AAm),poly-N-isopropylacrylamide, poly N-isopropylpolyacrylamide, sulfobetaineacrylate (SBA), carboxybetaine acrylate (CBA), phosphorylcholineacrylate (PCA), sulfobetaine methacrylate (SBMA), carboxybetainemethacrylate (CBMA), phosphorylcholine methacrylate (PCMA), polyethyleneglycol acrylate, methacrylate, polyethylene glycol(PEG)-thiol/PEG-acrylate, acrylamide/N,N′-bis(acryloyl)cystamine (BACy),PEG/polypropylene oxide (PPO), polyacrylic acid, poly(hydroxyethylmethacrylate) (PHEMA), poly(methyl methacrylate) (PMMA),poly(N-isopropylacrylamide) (PNIPAAm), poly(lactic acid) (PLA),poly(lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL),poly(vinylsulfonic acid) (PVSA), poly(L-aspartic acid), poly(L-glutamicacid), polylysine, agar, agarose, alginate, heparin, alginate sulfate,dextran sulfate, hyaluronan, pectin, carrageenan, gelatin, chitosan,cellulose, collagen, glicydyl methacrylate (GMA),hydroxyethylmethacrylate (HEMA), hydroxyethylacrylate (HEA),hydroxypropylmethacrylate (HPMA), polyethylene glycol methacrylate(PEGMA), polyethylene glycol acrylate (PEGA), isocyanatoethylmethacrylate (IEM), or a copolymer thereof.

Embodiment P-8. The array of Embodiment P-5, wherein the particle shellcomprises polymerized units of polyacrylamide (AAm), glicydylmethacrylate (GMA), polyethylene glycol methacrylate (PEGMA),polyethylene glycol methacrylate (PEGMA), isocyanatoethyl methacrylate(IEM), or a copolymer thereof.

Embodiment P-9. The array of Embodiment P-5, wherein the particle shellcomprises polymerized units of a) polyethylene glycol methacrylate(PEGMA) and glicydyl methacrylate (GMA) or b) polyethylene glycolmethacrylate (PEGMA) and isocyanatoethyl methacrylate (IEM).

Embodiment P-10. The array of any one of Embodiments P-5 to P-9, whereinthe particle shell is permeable to a polymerase.

Embodiment P-11. The array of any one of Embodiments P-1 to P-4, whereinthe particle is a polymer particle comprising polymerized units ofpolyacrylamide (AAm), poly-N-isopropylacrylamide, polyN-isopropylpolyacrylamide, sulfobetaine acrylate (SBA), carboxybetaineacrylate (CBA), phosphorylcholine acrylate (PCA), sulfobetainemethacrylate (SBMA), carboxybetaine methacrylate (CBMA),phosphorylcholine methacrylate (PCMA), polyethylene glycol acrylate,methacrylate, polyethylene glycol (PEG)-thiol/PEG-acrylate,acrylamide/N,N′-bis(acryloyl)cystamine (BACy), PEG/polypropylene oxide(PPO), polyacrylic acid, poly(hydroxyethyl methacrylate) (PHEMA),poly(methyl methacrylate) (PMMA), poly(N-isopropylacrylamide) (PNIPAAm),poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA),polycaprolactone (PCL), poly(vinylsulfonic acid) (PVSA), poly(L-asparticacid), poly(L-glutamic acid), polylysine, agar, agarose, alginate,heparin, alginate sulfate, dextran sulfate, hyaluronan, pectin,carrageenan, gelatin, chitosan, cellulose, collagen, glicydylmethacrylate (GMA), hydroxyethylmethacrylate (HEMA),hydroxyethylacrylate (HEA), hydroxypropylmethacrylate (HPMA),polyethylene glycol methacrylate (PEGMA), polyethylene glycol acrylate(PEGA), isocyanatoethyl methacrylate (IEM), or a copolymer thereof.

Embodiment P-12. The array of any one of Embodiments P-1 to P-4, whereinthe particle is a polymer particle comprising polyacrylamide (AAm),glicydyl methacrylate (GMA), polyethylene glycol methacrylate (PEGMA),polyethylene glycol acrylate (PEGA), isocyanatoethyl methacrylate (IEM),or a copolymer thereof.

Embodiment P-13. The array of any one of Embodiments P-1 to P-4, whereinthe particle is a polymer particle comprising polymerized units ofpolyacrylamide (AAm) and glicydyl methacrylate (GMA); polyacrylamide(AAm) and isocyanatoethyl methacrylate (IEM); or glicydyl methacrylate(GMA).

Embodiment P-14. The array of any one of Embodiments P-11 to P-13,wherein the polymer particle is permeable to a polymerase.

Embodiment P-15. The array of any one of Embodiments P-1 to P-14,wherein the solid support comprises a polymer layer, wherein the polymerlayer comprises an amphiphilic copolymer.

Embodiment P-16. The array of any one of Embodiments P-1 to P-14,wherein the solid support comprises a polymer layer, wherein the polymerlayer comprises an amphiphilic acrylate copolymer or amphiphilicmethacrylate copolymer.

Embodiment P-17. The array of any one of Embodiments P-1 to P-16,wherein the solid support comprises a photoresist and polymer layer,wherein the photoresist is between the solid support and the polymerlayer.

Embodiment P-18. The array of any one of Embodiments P-1 to P-17,wherein the amphiphilic copolymer comprises a poloxamer.

Embodiment P-19. The array of any one of Embodiments P-1 to P-17,wherein the amphiphilic copolymer comprises a brush copolymer or a combpolymer.

Embodiment P-20. The array of any one of Embodiments P-1 to P-17,wherein the amphiphilic copolymer comprises polymerized units ofalkoxysilyl polymers.

Embodiment P-21. The array of any one of Embodiments P-15 to P-20,wherein amphiphilic copolymer comprises polymerized units of3-(trimethoxysilyl)propyl methacrylate (TMSPM),3-(trimethoxysilyl)propyl methacrylate (TMSPA), 3-(triethoxysilyl)propylmethacrylate (TESPM), 3-(triethoxysilyl)propyl acrylate (TESPA),3-(dimethoxy(1-methylethoxy)silyl]propyl methacrylate,3-(ethoxydimethoxysilyl)propyl 2-methyl-2-propenoate,3-(Tripropoxysilyl)propyl 2-methyl-2-propenoate,2-Methyl-3-(triethoxysilyl)propyl 2-methyl-2-propenoate,3-(Methyldipropoxysilyl)propyl 2-methyl-2-propenoate,3-(Diethoxymethylsilyl)propyl 2-methyl-2-propenoate,3-[Diethoxy(2-hydroxyethoxy)silyl]propyl 2-methyl-2-propenoate,3-(Butyldimethoxysilyl)propyl 2-methyl-2-propenoate.

Embodiment P-22. The array of Embodiment P-20 or P-21, furthercomprising polymerized units of polyethylene glycol methacrylate(PEGMA), polyethylene glycol acrylate (PEGA), sulfobetaine acrylate(SBA), carboxybetaine acrylate (CBA), phosphorylcholine acrylate (PCA),sulfobetaine methacrylate (SBMA), carboxybetaine methacrylate (CBMA), orphosphorylcholine methacrylate (PCMA).

Embodiment P-23. The array of any one of Embodiments P-15 to P-20,wherein amphiphilic copolymer comprises polymerized units of alkoxysilylpolymers and polymerized units of polyethylene glycol methacrylate(PEGMA), or polyethylene glycol acrylate (PEGA).

Embodiment P-24. The array of any one of Embodiments P-15 to P-20,wherein amphiphilic copolymer comprises polymerized units of3-(trimethoxysilyl)propyl methacrylate (TMSPM),3-(trimethoxysilyl)propyl methacrylate (TMSPA) and polymerized units ofpolyethylene glycol methacrylate (PEGMA), or polyethylene glycolacrylate (PEGA).

Embodiment P-25. The array of any one of Embodiments P-1 to P-22,wherein the average longest dimension of the particle is from about 150nm to about 600 nm.

Embodiment P-26. The array of any one of Embodiments P-1 to P-22,wherein the average longest dimension of the particle is from about 350nm to about 600 nm.

Embodiment P-27. The array of any one of Embodiments P-1 to P-22,wherein the average longest dimension of the particle is from about 400nm to about 500 nm.

Embodiment P-28. The array of any one of Embodiments P-1 to P-27,wherein the wells are separated from each other by about 0.2 μm to about2.0 μm.

Embodiment P-29. The array of any one of Embodiments P-1 to P-27,wherein the wells are separated from each other by about 0.7 μm to about1.5 μm.

Embodiment P-30. The array of any one of Embodiments P-1 to P-29,wherein the wells are from about 0.2 μm to about 2 μm in diameter, andwherein the wells are about 0.5 μm to about 2 μm in depth.

Embodiment P-31. The array of any one of Embodiments P-1 to P-30,wherein greater than 50%, 60%, 70%, 80%, 90% or 95% of the wellscomprise a particle.

Embodiment P-32. The array of any one of Embodiments P-1 to P-30,wherein greater than 90% of the wells contain a particle.

Embodiment P-33. The array of any one of Embodiments P-1 to P-30,wherein greater than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%of the wells comprise a particle.

Embodiment P-34. The array of any one of Embodiments P-31 to P-33,wherein each of the particles comprise oligonucleotide moietiessubstantially identical to all the particles in the array.

Embodiment P-35. The array of any one of Embodiments P-31 to P-33,wherein each of the particles comprise at least two species ofoligonucleotide moieties that are substantially identical to all theparticles in the array.

Embodiment P-36. The array of any one of Embodiments P-1 to P-34,wherein the interstitial regions are substantially free ofoligonucleotide moieties.

Embodiment P-37. The array of any one of Embodiments P-1 to P-36,wherein each of the plurality of bioconjugate reactive moieties comprisean amine moiety, aldehyde moiety, alkyne moiety, azide moiety,carboxylic acid moiety, dibenzocyclooctyne (DBCO) moiety, tetrazinemoiety, epoxy moiety, isocyanate moiety, furan moiety, maleimide moiety,thiol moiety, or transcyclooctene (TCO) moiety.

Embodiment P-38. The array of any one of Embodiments P-1 to P-36,wherein each of the plurality of bioconjugate reactive moieties comprisean amine moiety, azide moiety, alkyne moiety, dibenzocyclooctyne (DBCO)moiety, epoxy moiety, or isocyanate moiety.

Embodiment P-39. The array of any one of Embodiments P-1 to P-38,wherein the oligonucleotide moiety is about 5 to about 40 nucleotides inlength.

Embodiment P-40. The array of any one of Embodiments P-1 to P-38,wherein the oligonucleotide moiety is about 20 to about 30 nucleotidesin length.

Embodiment P-41. The array of any one of Embodiments P-1 to P-39,wherein the oligonucleotide moiety is capable of hybridizing to acomplementary sequence of a template nucleic acid.

Embodiment P-42. The array of any one of Embodiments P-1 to P-41,wherein the particle comprises a plurality of azide moieties, alkynemoieties, dibenzocyclooctyne (DBCO) moieties, epoxy moieties, orisocyanate moieties.

Embodiment P-43. The array of any one of Embodiments P-1 to P-41,wherein the particle comprises a plurality of oligonucleotide moieties.

Embodiment P-44. A method of amplifying a target polynucleotide, themethod comprising: contacting the array of any one of Embodiments P-1 toP-43 with a plurality of oligonucleotide moieties, each oligonucleotidemoiety comprising a bioconjugate reactive moiety that reacts and forms abioconjugate linker that covalently links the oligonucleotide moiety tothe particle, contacting the array with a sample comprising a targetpolynucleotide; and amplifying the target polynucleotide to produce anamplification product, wherein amplifying comprises extension of theoligonucleotide moiety hybridized to the target polynucleotide.

Embodiment P-45. A method of amplifying a target polynucleotide, themethod comprising: contacting the array of Embodiment P-43 with a samplecomprising a target polynucleotide; and amplifying the targetpolynucleotide to produce an amplification product, wherein amplifyingcomprises extension of the oligonucleotide moiety hybridized to thetarget polynucleotide.

Embodiment P-46. A method of making an array of nucleic acids on asurface, the method comprising: a) providing a solid support comprisinga surface, the surface comprising a plurality of wells wherein the wellsare separated from each other by interstitial regions on the surface; b)providing a plurality of particles, wherein each particle comprises aplurality of bioconjugate reactive moieties; c) arraying the particlesonto the surface; d) contacting the particles with a plurality ofoligonucleotide moieties, wherein each oligonucleotide moiety comprisesa bioconjugate reactive moiety that reacts and forms a bioconjugatelinker that covalently links the oligonucleotide moiety to the particle.

Embodiment P-47. A method of making an array of template nucleic acidson a surface, the method comprising: a) providing a solid supportcomprising a surface, the surface comprising a plurality of wellswherein the wells are separated from each other by interstitial regionson the surface; b) providing a plurality of particles, wherein eachparticle comprises a plurality of oligonucleotide moieties; wherein theaverage longest dimension of the particle is from about 150 nm to about1,000 nm; c) arraying the particles onto the surface; d) contacting theparticles with a plurality of template nucleic acid moieties, wherein acomplementary sequence of the template nucleic acid moieties hybridizesto the oligonucleotide moiety of the particle.

Embodiment P-48. The method of Embodiment P-46 or P-47, wherein theparticle is a functionalized particle comprising a particle core and aparticle shell, wherein said particle shell comprises the plurality ofbioconjugate reactive moieties, the plurality of oligonucleotidemoieties, or a combination thereof, wherein each of said bioconjugatereactive moieties and each of said oligonucleotide moieties comprise alinker binding said bioconjugate reactive moieties and oligonucleotideto said particle core.

Embodiment P-49. The method of Embodiment P-46 or P-47, wherein theparticle is a polymer particle.

Embodiment P-50. The method of Embodiment P-49, wherein arraying theparticles comprises contacting the surface with a first solutioncomprising the plurality of particles in an anti-solvent.

Embodiment P-51. The method of Embodiment P-50, wherein the particlecomprises acrylamide and the anti-solvent is an aqueous ethanolsolution.

Embodiment P-52. The method of Embodiment P-50, wherein the particlecomprises sulfobetaine acrylate (SBA), carboxybetaine acrylate (CBA),phosphorylcholine acrylate (PCA), sulfobetaine methacrylate (SBMA),carboxybetaine methacrylate (CBMA), or phosphorylcholine methacrylate(PCMA) and the anti-solvent is an aqueous acetone solution.

Embodiment P-53. The method of Embodiment P-50, further comprisingremoving the first solution and contacting the surface with a secondsolution, wherein the second solution is an aqueous solution capable ofexpanding the volume of the particle.

Embodiment P-54. A nucleic acid sequencing device, comprising: a stageconfigured to hold an array of any one of Embodiments P-1 to P-43; anarray of any one of Embodiments P-1 to P-43; and a detector forobtaining sequencing data.

ADDITIONAL EMBODIMENTS

Embodiment 1. A solid support comprising two or more wells, wherein eachwell is separated by about 0.2 μm to about 2.0 μm and each wellcomprises at least one particle, said particle comprising a plurality ofoligonucleotide moieties covalently attached to said particle via abioconjugate linker, wherein the bioconjugate linker is formed via areaction between a particle polymer comprising a first bioconjugatereactive moiety and an oligonucleotide comprising a second bioconjugatereactive moiety, and wherein the average longest dimension of theparticle is from about 100 nm to about 1000 nm.

Embodiment 2. The solid support of Embodiment 1, wherein the solidsupport comprises a polymer layer.

Embodiment 3. The solid support of Embodiment 2, wherein the polymerlayer comprises polymerized units of alkoxysilyl methacrylate,alkoxysilyl acrylate, alkoxysilyl methylacrylamide, alkoxysilylmethylacrylamide, or a copolymer thereof.

Embodiment 4. The solid support of Embodiment 1, wherein the solidsupport comprises a photoresist, wherein the photoresist is asilsesquioxane resist, an epoxy-based polymer resist,poly(vinylpyrrolidone-vinyl acrylic acid) copolymer resist, anOff-stoichiometry thiol-enes (OSTE) resist, amorphous fluoropolymerresist, a crystalline fluoropolymer resist, polysiloxane resist, or aorganically modified ceramic polymer resist.

Embodiment 5. The solid support of any one of Embodiments 1 to 4,wherein the particle comprises silica, glass, ceramic, metal, magneticmaterial, or a paramagnetic material.

Embodiment 6. The solid support of any one of Embodiments 1 to 5,wherein the particle polymer comprises polymerized units ofpolyacrylamide (AAm), poly-N-isopropylacrylamide, polyN-isopropylpolyacrylamide, sulfobetaine acrylate (SBA), carboxybetaineacrylate (CBA), phosphorylcholine acrylate (PCA), sulfobetainemethacrylate (SBMA), carboxybetaine methacrylate (CBMA),phosphorylcholine methacrylate (PCMA), polyethylene glycol acrylate,methacrylate, N-vinyl pyrrolidone, polyethylene glycol(PEG)-thiol/PEG-acrylate, acrylamide/N,N′-bis(acryloyl)cystamine (BACy),PEG/polypropylene oxide (PPO), polyacrylic acid, poly(hydroxyethylmethacrylate) (PHEMA), poly(methyl methacrylate) (PMMA),poly(N-isopropylacrylamide) (PNIPAAm), poly(lactic acid) (PLA),poly(lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL),poly(vinylsulfonic acid) (PVSA), poly(L-aspartic acid), poly(L-glutamicacid), polylysine, agar, agarose, alginate, heparin, alginate sulfate,dextran sulfate, hyaluronan, pectin, carrageenan, gelatin, chitosan,cellulose, collagen, glicydyl methacrylate (GMA), glicydyl methacrylate(GMA) azide, hydroxyethylmethacrylate (HEMA), hydroxyethylacrylate(HEA), hydroxypropylmethacrylate (HPMA), polyethylene glycolmethacrylate (PEGMA), polyethylene glycol acrylate (PEGA),isocyanatoethyl methacrylate (IEM), or a copolymer thereof.

Embodiment 7. The solid support of any one of Embodiments 1 to 4,wherein the particle polymer comprises polymerized units ofpolyacrylamide (AAm), glicydyl methacrylate (GMA), glicydyl methacrylate(GMA) azide, polyethylene glycol methacrylate (PEGMA), polyethyleneglycol methacrylate (PEGMA), isocyanatoethyl methacrylate (IEM), or acopolymer thereof.

Embodiment 8. The solid support of any one of Embodiments 1 to 4,wherein the particle polymer comprises polymerized units of a)polyethylene glycol methacrylate (PEGMA) and glicydyl methacrylate(GMA), b) polyethylene glycol methacrylate (PEGMA) and isocyanatoethylmethacrylate (IEM), or c) polyethylene glycol methacrylate (PEGMA) andglicydyl methacrylate (GMA) azide.

Embodiment 9. The solid support of any one of Embodiments 1 to 4,wherein the particle is a polymeric particle comprising polymerizedunits of polyacrylamide (AAm), poly-N-isopropylacrylamide, polyN-isopropylpolyacrylamide, sulfobetaine acrylate (SBA), carboxybetaineacrylate (CBA), phosphorylcholine acrylate (PCA), sulfobetainemethacrylate (SBMA), carboxybetaine methacrylate (CBMA),phosphorylcholine methacrylate (PCMA), polyethylene glycol acrylate,methacrylate, polyethylene glycol (PEG)-thiol/PEG-acrylate,acrylamide/N,N′-bis(acryloyl)cystamine (BACy), PEG/polypropylene oxide(PPO), polyacrylic acid, poly(hydroxyethyl methacrylate) (PHEMA),poly(methyl methacrylate) (PMMA), poly(N-isopropylacrylamide) (PNIPAAm),poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA),polycaprolactone (PCL), poly(vinylsulfonic acid) (PVSA), poly(L-asparticacid), poly(L-glutamic acid), polylysine, agar, agarose, alginate,heparin, alginate sulfate, dextran sulfate, hyaluronan, pectin,carrageenan, gelatin, chitosan, cellulose, collagen, glicydylmethacrylate (GMA), hydroxyethylmethacrylate (HEMA),hydroxyethylacrylate (HEA), hydroxypropylmethacrylate (HPMA),polyethylene glycol methacrylate (PEGMA), polyethylene glycol acrylate(PEGA), isocyanatoethyl methacrylate (IEM), or a copolymer thereof.

Embodiment 10. The solid support of any one of Embodiments 1 to 4,wherein the particle is a polymeric particle comprising polyacrylamide(AAm), glicydyl methacrylate (GMA), polyethylene glycol methacrylate(PEGMA), polyethylene glycol acrylate (PEGA), isocyanatoethylmethacrylate (IEM), or a copolymer thereof.

Embodiment 11. The solid support of any one of Embodiments 1 to 4,wherein the particle is a polymeric particle comprising polymerizedunits of polyacrylamide (AAm) and glicydyl methacrylate (GMA);polyacrylamide (AAm) and isocyanatoethyl methacrylate (IEM); or glicydylmethacrylate (GMA).

Embodiment 12. The solid support of any one of Embodiments 9 to 11,wherein the polymeric particle is permeable to a polymerase.

Embodiment 13. The solid support of any one of Embodiments 1 to 12,wherein the solid support further comprises an amphiphilic copolymerlayer.

Embodiment 14. The solid support of Embodiment 13, wherein theamphiphilic copolymer comprises a poloxamer.

Embodiment 15. The solid support of Embodiment 1, wherein theamphiphilic copolymer comprises a brush copolymer or a comb polymer.

Embodiment 16. The solid support of Embodiment 13, wherein theamphiphilic copolymer comprises polymerized units of alkoxysilylpolymers.

Embodiment 17. The solid support of Embodiment 13, wherein amphiphiliccopolymer comprises polymerized units of 3-(trimethoxysilyl)propylmethacrylate (TMSPM), 3-(trimethoxysilyl)propyl methacrylate (TMSPA),3-(triethoxysilyl)propyl methacrylate (TESPM), 3-(triethoxysilyl)propylacrylate (TESPA), hydroxyethylmethacrylate (HEMA),3-(dimethoxy(1-methylethoxy)silyl]propyl methacrylate,3-(ethoxydimethoxysilyl)propyl 2-methyl-2-propenoate,3-(Tripropoxysilyl)propyl 2-methyl-2-propenoate,2-Methyl-3-(triethoxysilyl)propyl 2-methyl-2-propenoate,3-(Methyldipropoxysilyl)propyl 2-methyl-2-propenoate,3-(Diethoxymethylsilyl)propyl 2-methyl-2-propenoate,3-[Diethoxy(2-hydroxyethoxy)silyl]propyl 2-methyl-2-propenoate,3-(Butyldimethoxysilyl)propyl 2-methyl-2-propenoate.

Embodiment 18. The solid support of Embodiment 16 or 17, furthercomprising polymerized units of polyethylene glycol methacrylate(PEGMA), polyethylene glycol acrylate (PEGA), sulfobetaine acrylate(SBA), carboxybetaine acrylate (CBA), phosphorylcholine acrylate (PCA),sulfobetaine methacrylate (SBMA), carboxybetaine methacrylate (CBMA), orphosphorylcholine methacrylate (PCMA).

Embodiment 19. The solid support of Embodiment 13, wherein amphiphiliccopolymer comprises polymerized units of alkoxysilyl polymers andpolymerized units of polyethylene glycol methacrylate (PEGMA), orpolyethylene glycol acrylate (PEGA).

Embodiment 20. The solid support of Embodiment 13, wherein amphiphiliccopolymer comprises polymerized units of 3-(trimethoxysilyl)propylmethacrylate (TMSPM), 3-(trimethoxysilyl)propyl methacrylate (TMSPA) andpolymerized units of polyethylene glycol methacrylate (PEGMA), orpolyethylene glycol acrylate (PEGA).

Embodiment 21. The solid support of any one of Embodiments 1 to 20,wherein the average longest dimension of the particle is from about 150nm to about 600 nm.

Embodiment 22. The solid support of any one of Embodiments 1 to 20,wherein the average longest dimension of the particle is from about 350nm to about 600 nm.

Embodiment 23. The solid support of any one of Embodiments 1 to 20,wherein the average longest dimension of the particle is from about 400nm to about 500 nm.

Embodiment 24. The solid support of any one of Embodiments 1 to 20,wherein the average longest dimension of the particle is about 450 nm.

Embodiment 25. The solid support of any one of Embodiments 1 to 24,wherein the solid support comprises about 1×10⁶ to about 5×10¹⁰ wells.

Embodiment 26. The solid support of any one of Embodiments 1 to 25,wherein the wells are separated from each other by about 0.5 μm to about2.0 μm.

Embodiment 27. The solid support of any one of Embodiments 1 to 25,wherein the wells are separated from each other by about 0.7 μm to about1.5 μm.

Embodiment 28. The solid support of any one of Embodiments 1 to 25,wherein the wells are from about 0.2 μm to about 2 μm in diameter, andwherein the wells are about 0.5 μm to about 2 μm in depth.

Embodiment 29. The solid support of any one of Embodiments 25 to 28,wherein greater than 50%, 60%, 70%, 80%, 90% or 95% of the wellscomprise a particle.

Embodiment 30. The solid support of any one of Embodiments 25 to 28,wherein greater than 90% of the wells contain a particle.

Embodiment 31. The solid support of any one of Embodiments 25 to 28,wherein greater than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%of the wells comprise a particle.

Embodiment 32. The solid support of any one of Embodiments 1 to 31,wherein each particle comprises substantially the same oligonucleotidemoieties.

Embodiment 33. The solid support of any one of Embodiments 1 to 31,wherein each of the particles comprise at least two populations ofsubstantially the same oligonucleotide moieties.

Embodiment 34. The solid support of any one of Embodiments 1 to 33,wherein the first bioconjugate reactive moiety is an amine moiety,aldehyde moiety, alkyne moiety, azide moiety, carboxylic acid moiety,dibenzocyclooctyne (DBCO) moiety, tetrazine moiety, epoxy moiety,isocyanate moiety, furan moiety, maleimide moiety, thiol moiety, ortranscyclooctene (TCO) moiety.

Embodiment 35. The solid support of any one of Embodiments 1 to 33,wherein the first bioconjugate reactive moiety is an amine moiety, azidemoiety, alkyne moiety, dibenzocyclooctyne (DBCO) moiety, epoxy moiety,or isocyanate moiety.

Embodiment 36. The solid support of any one of Embodiments 1 to 35,wherein the oligonucleotide moiety is about 5 to about 40 nucleotides inlength.

Embodiment 37. The solid support of any one of Embodiments 1 to 35,wherein the oligonucleotide moiety is about 20 to about 35 nucleotidesin length.

Embodiment 38. The solid support of any one of Embodiments 1 to 37,wherein the oligonucleotide moiety is capable of hybridizing to acomplementary sequence of a template nucleic acid.

Embodiment 39. The solid support of any one of Embodiments 1 to 37,wherein the oligonucleotide moiety is capable of hybridizing to a commonsequence in a library of nucleic acid molecules.

Embodiment 40. A method of amplifying a target polynucleotide, themethod comprising: contacting the solid support of any one ofEmbodiments 1 to 39 with a sample comprising a target polynucleotide;and amplifying the target polynucleotide to produce an amplificationproduct, wherein amplifying comprises extension of the oligonucleotidemoiety hybridized to the target polynucleotide.

Embodiment 41. A method of making an array of template nucleic acids,the method comprising: contacting a solid support comprising two or morewells with a plurality of particles, wherein each particle comprises aplurality of oligonucleotide moieties attached to said particle via abioconjugate linker; wherein the average longest dimension of theparticle is from about 150 nm to about 1,000 nm; and contacting saidparticles with a plurality of template nucleic acid moieties, wherein acomplementary sequence of the template nucleic acid moieties hybridizesto the oligonucleotide moiety of the particle and is extended with apolymerase to form an array of template nucleic acids.

1. A solid support comprising two or more wells, wherein each well isseparated by about 0.2 μm to about 2.0 μm and each well comprises atleast one particle, said particle comprising a plurality ofoligonucleotide moieties covalently attached to said particle via abioconjugate linker, wherein the bioconjugate linker is formed via areaction between a particle polymer comprising a first bioconjugatereactive moiety and an oligonucleotide comprising a second bioconjugatereactive moiety, and wherein the average longest dimension of theparticle is from about 100 nm to about 1000 nm, wherein said solidsupport comprises a polymer layer comprising polymerized units ofalkoxysilyl methacrylate, alkoxysilyl acrylate, alkoxysilylmethylacrylamide, alkoxysilyl methylacrylamide, or a copolymer thereof.2. (canceled)
 3. (canceled)
 4. The solid support of claim 1, wherein thesolid support comprises a photoresist, wherein the photoresist is asilsesquioxane resist, an epoxy-based polymer resist,poly(vinylpyrrolidone-vinyl acrylic acid) copolymer resist, anOff-stoichiometry thiol-enes (OSTE) resist, amorphous fluoropolymerresist, a crystalline fluoropolymer resist, polysiloxane resist, or aorganically modified ceramic polymer resist.
 5. The solid support ofclaim 1, wherein the particle comprises silica, glass, ceramic, metal,magnetic material, or a paramagnetic material.
 6. The solid support ofclaim 1, wherein the particle polymer comprises polymerized units ofpolyacrylamide (AAm), poly-N-isopropylacrylamide, polyN-isopropylpolyacrylamide, sulfobetaine acrylate (SBA), carboxybetaineacrylate (CBA), phosphorylcholine acrylate (PCA), sulfobetainemethacrylate (SBMA), carboxybetaine methacrylate (CBMA),phosphorylcholine methacrylate (PCMA), polyethylene glycol acrylate,methacrylate, polyethylene glycol (PEG)-thiol/PEG-acrylate,acrylamide/N,N′-bis(acryloyl)cystamine (BACy), PEG/polypropylene oxide(PPO), polyacrylic acid, poly(hydroxyethyl methacrylate) (PHEMA),poly(methyl methacrylate) (PMMA), poly(N-isopropylacrylamide) (PNIPAAm),poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA),polycaprolactone (PCL), poly(vinylsulfonic acid) (PVSA), poly(L-asparticacid), poly(L-glutamic acid), polylysine, agar, agarose, alginate,heparin, alginate sulfate, dextran sulfate, hyaluronan, pectin,carrageenan, gelatin, chitosan, cellulose, collagen, glicydylmethacrylate (GMA), glicydyl methacrylate (GMA) azide,hydroxyethylmethacrylate (HEMA), hydroxyethylacrylate (HEA),hydroxypropylmethacrylate (HPMA), polyethylene glycol methacrylate(PEGMA), polyethylene glycol acrylate (PEGA), isocyanatoethylmethacrylate (IEM), or a copolymer thereof.
 7. The solid support ofclaim 1, wherein the particle polymer comprises polymerized units ofpolyacrylamide (AAm), glicydyl methacrylate (GMA), glicydyl methacrylate(GMA) azide, polyethylene glycol methacrylate (PEGMA), polyethyleneglycol methacrylate (PEGMA), isocyanatoethyl methacrylate (IEM), or acopolymer thereof.
 8. The solid support of claim 1, wherein the particlepolymer comprises polymerized units of a) polyethylene glycolmethacrylate (PEGMA) and glicydyl methacrylate (GMA), b) polyethyleneglycol methacrylate (PEGMA) and isocyanatoethyl methacrylate (IEM), orc) polyethylene glycol methacrylate (PEGMA) and glicydyl methacrylate(GMA) azide.
 9. The solid support of claim 1, wherein the particle is apolymeric particle comprising polymerized units of polyacrylamide (AAm),poly-N-isopropylacrylamide, poly N-isopropylpolyacrylamide, sulfobetaineacrylate (SBA), carboxybetaine acrylate (CBA), phosphorylcholineacrylate (PCA), sulfobetaine methacrylate (SBMA), carboxybetainemethacrylate (CBMA), phosphorylcholine methacrylate (PCMA), polyethyleneglycol acrylate, methacrylate, polyethylene glycol(PEG)-thiol/PEG-acrylate, acrylamide/N,N′-bis(acryloyl)cystamine (BACy),PEG/polypropylene oxide (PPO), polyacrylic acid, poly(hydroxyethylmethacrylate) (PHEMA), poly(methyl methacrylate) (PMMA),poly(N-isopropylacrylamide) (PNIPAAm), poly(lactic acid) (PLA),poly(lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL),poly(vinylsulfonic acid) (PVSA), poly(L-aspartic acid), poly(L-glutamicacid), polylysine, agar, agarose, alginate, heparin, alginate sulfate,dextran sulfate, hyaluronan, pectin, carrageenan, gelatin, chitosan,cellulose, collagen, glicydyl methacrylate (GMA),hydroxyethylmethacrylate (HEMA), hydroxyethylacrylate (HEA),hydroxypropylmethacrylate (HPMA), polyethylene glycol methacrylate(PEGMA), polyethylene glycol acrylate (PEGA), isocyanatoethylmethacrylate (IEM), or a copolymer thereof. 10.-12. (canceled)
 13. Thesolid support of claim 1, wherein the solid support further comprises anamphiphilic copolymer layer.
 14. The solid support of claim 13, whereinthe amphiphilic copolymer comprises a poloxamer.
 15. The solid supportof claim 13, wherein the amphiphilic copolymer comprises a brushcopolymer or a comb polymer. 16.-20. (canceled)
 21. The solid support ofclaim 1, wherein the average longest dimension of the particle is fromabout 150 nm to about 600 nm.
 22. The solid support of claim 1, whereinthe average longest dimension of the particle is from about 350 nm toabout 600 nm.
 23. The solid support of claim 1, wherein the averagelongest dimension of the particle is from about 400 nm to about 500 nm.24. (canceled)
 25. The solid support of claim 1, wherein the solidsupport comprises about 1×10⁵ to about 5×10¹⁰ wells.
 26. The solidsupport of claim 1, wherein the wells are separated from each other byabout 0.5 μm to about 2.0 μm.
 27. (canceled)
 28. The solid support ofclaim 1, wherein the wells are from about 0.2 μm to about 2 μm indiameter, and wherein the wells are about 0.5 μm to about 2 μm in depth.29. The solid support of claim 25, wherein greater than 50%, 60%, 70%,80%, 90% or 95% of the wells comprise a particle. 30.-32. (canceled) 33.The solid support of claim 1, wherein each of the particles comprise atleast two populations of substantially the same oligonucleotidemoieties. 34.-37. (canceled)
 38. The solid support of claim 1, whereineach of the oligonucleotide moieties is capable of hybridizing to acomplementary sequence of a template nucleic acid.
 39. (canceled)
 40. Amethod of amplifying a target polynucleotide, the method comprising:contacting the solid support of claim 1 with a sample comprising atarget polynucleotide; and amplifying the target polynucleotide toproduce an amplification product, wherein amplifying compriseshybridizing an oligonucleotide moiety of said plurality ofoligonucleotide moieties and extending the oligonucleotide moietyhybridized to the target polynucleotide.
 41. (canceled)